Antigen binding molecules comprising a TNF family ligand trimer and a Tenascin binding moiety

ABSTRACT

The invention relates to novel TNF family ligand trimer-containing antigen binding molecules comprising (a) at least one antigen binding moiety capable of specific binding to Tenascin-C (TnC) and (b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecules are characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof. The invention further relates to methods of producing these molecules and to methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No. PCT/EP2017/060870, filed May 8, 2017, which claims priority from European Patent Application No. 16169244.7, filed May 11, 2016. The contents of each of the foregoing applications are incorporated herein by reference in their entireties.

SEQUENCE LISTING

This application contains a Sequence Listing submitted via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 20, 2018, is named P33586-US_SL.txt and is 538,620 bytes in size.

FIELD OF THE INVENTION

The invention relates to novel TNF family ligand trimer-containing antigen binding molecules comprising (a) at least one antigen binding moiety capable of specific binding to Tenascin-C (TnC) and (b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecules are characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof. The invention further relates to methods of producing these molecules and to methods of using the same.

BACKGROUND

Ligands interacting with molecules of the TNF (tumor necrosis factor) receptor superfamily have pivotal roles in the organization and function of the immune system. While regulating normal functions such as immune responses, hematopoiesis and morphogenesis, the TNF family ligands (also called cytokines) play a role in tumorgenesis, transplant rejection, septic shock, viral replication, bone resorption, rheumatoid arthritis and diabetes (Aggarwal, 2003). The TNF ligand family comprises 18 genes encoding 19 type II (i.e. intracellular N terminus and extracellular C-terminus) transmembrane proteins, characterized by the presence of a conserved C-terminal domain coined the ‘TNF homology domain’ (THD). This domain is responsible for receptor binding and is thus critical for the biological activity of the TNF ligand family members. The sequence identity between family members is about 20-30% (Bodmer, 2002). Members of the TNF ligand family exert their biological function as self-assembling, noncovalent trimers (Banner et al, Cell 1993, 73, 431-445). Thus, the TNF family ligands form a trimer that is able to bind to and to activate the corresponding receptors of TNFR superfamily.

4-1BB (CD137), a member of the TNF receptor superfamily, has been first identified as a molecule whose expression is induced by T-cell activation (Kwon and Weissman, 1989). Subsequent studies demonstrated expression of 4-1BB in T- and B-lymphocytes (Snell et al., 2011; Zhang et al., 2010), NK-cells (Lin et al., 2008), NKT-cells (Kim et al., 2008), monocytes (Kienzle and von Kempis, 2000; Schwarz et al., 1995), neutrophils (Heinisch et al., 2000), mast (Nishimoto et al., 2005) and dendritic cells as well as cells of non-hematopoietic origin such as endothelial and smooth muscle cells (Broil et al., 2001; Olofsson et al., 2008). Expression of 4-1BB in different cell types is mostly inducible and driven by various stimulatory signals, such as T-cell receptor (TCR) or B-cell receptor triggering, as well as signaling induced through co-stimulatory molecules or receptors of pro-inflammatory cytokines (Diehl et al., 2002; von Kempis et al., 1997; Zhang et al., 2010).

Expression of 4-1BB ligand (4-1BBL or CD137L) is more restricted and is observed on professional antigen presenting cells (APC) such as B-cells, dendritic cells (DCs) and macrophages. Inducible expression of 4-1BBL is characteristic for T-cells, including both αβ and γδT-cell subsets, and endothelial cells (reviewed in Shao and Schwarz, 2011).

CD137 signaling is known to stimulate IFNγ secretion and proliferation of NK cells (Buechele et al., 2012; Lin et al., 2008; Melero et al., 1998) as well as to promote DC activation as indicated by their increased survival and capacity to secret cytokines and upregulate co-stimulatory molecules (Choi et al., 2009; Futagawa et al., 2002; Wilcox et al., 2002). However, CD137 is best characterized as a co-stimulatory molecule which modulates TCR-induced activation in both the CD4+ and CD8+ subsets of T-cells. In combination with TCR triggering, agonistic 4-1BB-specific antibodies enhance proliferation of T-cells, stimulate lymphokine secretion and decrease sensitivity of T-lymphocytes to activation-induced cells death (reviewed in Snell et al., 2011).

In line with these co-stimulatory effects of 4-1BB antibodies on T-cells in vitro, their administration to tumor bearing mice leads to potent anti-tumor effects in many experimental tumor models (Melero et al., 1997; Narazaki et al., 2010). However, 4-1BB usually exhibits its potency as an anti-tumor agent only when administered in combination with other immunomodulatory compounds (Curran et al., 2011; Guo et al., 2013; Morales-Kastresana et al., 2013; Teng et al., 2009; Wei et al., 2013), chemotherapeutic reagents (Ju et al., 2008; Kim et al., 2009), tumor-specific vaccination (Cuadros et al., 2005; Lee et al., 2011) or radiotherapy (Shi and Siemann, 2006). In vivo depletion experiments demonstrated that CD8+ T-cells play the most critical role in anti-tumoral effect of 4-1BB-specific antibodies. However, depending on the tumor model or combination therapy, which includes anti-4-1BB, contributions of other types of cells such as DCs, NK-cells or CD4+ T-cells have been reported (Melero et al., 1997; Murillo et al., 2009; Narazaki et al., 2010; Stagg et al., 2011).

In addition to their direct effects on different lymphocyte subsets, 4-1BB agonists can also induce infiltration and retention of activated T-cells in the tumor through 4-1BB-mediated upregulation of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) on tumor vascular endothelium (Palazon et al., 2011).

4-1BB triggering may also reverse the state of T-cell anergy induced by exposure to soluble antigen that may contribute to disruption of immunological tolerance in the tumor microenvironment or during chronic infections (Wilcox et al., 2004).

It appears that the immunomodulatory properties of 4-1BB agonistic antibodies in vivo require the presence of the wild type Fc-portion on the antibody molecule thereby implicating Fc-receptor binding as an important event required for the pharmacological activity of such reagents as has been described for agonistic antibodies specific to other apoptosis-inducing or immunomodulatory members of the TNFR-superfamily (Li and Ravetch, 2011; Teng et al., 2009). However, systemic administration of 4-1BB-specific agonistic antibodies with the functionally active Fc domain also induces expansion of CD8+ T-cells associated with liver toxicity (Dubrot et al., 2010) that is diminished or significantly ameliorated in the absence of functional Fc-receptors in mice. In human clinical trials (ClinicalTrials.gov, NCT00309023), Fc-competent 4-1BB agonistic antibodies (BMS-663513) administered once every three weeks for 12 weeks induced stabilization of the disease in patients with melanoma, ovarian or renal cell carcinoma. However, the same antibody given in another trial (NCT00612664) caused grade 4 hepatitis leading to termination of the trial (Simeone and Ascierto, 2012).

Collectively, the available pre-clinical and clinical data clearly demonstrate that there is a high clinical need for effective 4-1BB agonists. However, new generation drug candidates should not only effectively engage 4-1BB on the surface of hematopoietic and endothelial cells but also be capable of achieving that through mechanisms other than binding to Fc-receptors in order to avoid uncontrollable side effects. The latter may be accomplished through preferential binding to tumor-specific or tumor-associated moieties.

Tenascins are a highly conserved family of large multimeric extracellular matrix (ECM) glycoproteins, which is found in vertebrates. Four Tenascin paralogues have been identified in mammals, termed Tenascin-C, Tenascin-R, Tenascin-X and Tenascin-W. Tenascin family proteins have a common primary structure, comprising N-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats and a C-terminal fibrinogen-like globular domain. Via an N-terminal oligomerization domain, individual subunits assemble into trimers or, as is the case for TnC, even hexamers. Indeed, the oligomerization domain of TnC was reported to improve trimerzation of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand when fused to the respective TNF receptor ligand (see Wyzgol et al., J Immunol. 183(3), 1851-61 (2009) and Berg et al., Cell Deatch and Differentiation 14(12, 2021-2034 (2007)). These studies used fragments of the TnC polypeptide chain to stabilice the active trimeric form of the TNF ligands in TnC-TNF-ligand fusion polypetides.

Mammalian Tenascin-C (TnC) monomers typically have 14.5 EGF-like repeats and 8 fibronectin type III domain repeats that are shared by all Tenascin-C isoforms. However, up to 9 additional fibronectin type III domain repeats (domains A1 to D) can be independently included or excluded by alternative splicing, giving rise to a large number of Tenascin-C isoforms (see e.g. Hsia and Schwarzbauer, J Biol Chem 280, 26641-26644 (2005)).

Tenascin-C is transiently expressed in the developing embryo, but virtually absent from adult tissues. It reappears, however, in tissues undergoing remodeling processes, including certain pathological conditions such as wound healing, inflammation and cancer (reviewed in Chiquet-Ehrismann & Chiquet, J Pathol 200, 488-499 (2003)).

Importantly, Tenascin-C is highly expressed in the majority of malignant solid tumors, including tumors of the brain, breast, colon, lung, skin and other organs (reviewed in Orend and Chiquet-Ehrismann, Cancer Letters 244, 143-163 (2006)), where it may be expressed by transformed epithelial cells as well as stromal cells in the tumor microenvironment (Yoshida et al., J Pathol 182, 421-428 (1997), Hanamura et al., Int J Cancer 73, 10-15 (1997)). In particular, the “large isoform” of Tenascin-C, containing the alternatively spliced domains A1 to D, is expressed in invasive carcinomas while being nearly undetectable in healthy adult tissues (Borsi et al., Int J Cancer 52, 688-692 (1992), Carnemolla et al., Eur J Biochem 205, 561-567 (1992)).

SUMMARY OF THE INVENTION

There remains a need for active and safe 4-1BB agonists. The bispecific molecules of the invention with at least one TnC antigen binding moiety targeting the molecules of the invention to tumor cells of a broad range of malignancies, and a trimeric and thus biologically active TNF ligand, provide a unique targeting tool to address tumor cells. The antigen binding molecules of the invention have sufficient stability to be pharmaceutically useful and can be used to treat different cancer with increased efficiency and safety.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to Tenascin-C (TnC) and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof.

In a particular aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof, and (c) an Fc domain composed of a first and a second subunit capable of stable association.

In a further aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that

-   -   (i) the first polypeptide contains a CH1 or CL domain and the         second polypeptide contains a CL or CH1 domain, respectively,         wherein the second polypeptide is linked to the first         polypeptide by a disulfide bond between the CH1 and CL domain,         and wherein the first polypeptide comprises two ectodomains of a         TNF ligand family member or fragments thereof that are connected         to each other and to the CH1 or CL domain by a peptide linker         and wherein the second polypeptide comprises one ectodomain of         said TNF ligand family member or a fragment thereof connected         via a peptide linker to the CL or CH1 domain of said         polypeptide, or     -   (ii) the first polypeptide contains a CH3 domain and the second         polypeptide contains a CH3 domain, respectively, and wherein the         first polypeptide comprises two ectodomains of a TNF ligand         family member or fragments thereof that are connected to each         other and to the C-terminus of the CH3 domain by a peptide         linker and wherein the second polypeptide comprises only one         ectodomain of said TNF ligand family member or a fragment         thereof connected via a peptide linker to C-terminus of the CH3         domain of said polypeptide, or     -   (iii) the first polypeptide contains a VH-CL or a VL-CH1 domain         and the second polypeptide contains a VL-CH1 domain or a VH-CL         domain, respectively, wherein the second polypeptide is linked         to the first polypeptide by a disulfide bond between the CH1 and         CL domain, and wherein the first polypeptide comprises two         ectodomains of a TNF ligand family member or fragments thereof         that are connected to each other and to to VH or VL by a peptide         linker and wherein the second polypeptide comprises one         ectodomain of said TNF ligand family member or a fragment         thereof connected via a peptide linker to VL or VH of said         polypeptide.

In a particular aspect, the TNF ligand family member is one that costimulates human T-cell activation. Thus, the TNF family ligand trimer-containing antigen binding molecule comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof, wherein the TNF ligand family member costimulates human T-cell activation. More particularly, the TNF ligand family member is selected from 4-1BBL and OX40L.

In one aspect, the TNF ligand family member is 4-1BBL.

In a further aspect, the ectodomain of a TNF ligand family member comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 183, SEQ ID NO: 192, SEQ ID NO: 193 and SEQ ID NO: 194, particularly the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 183.

In another aspect, the ectodomain of a TNF ligand family member or fragment thereof comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183, particularly the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 183. More particularly, the ectodomain of a TNF ligand family member comprises the amino acid sequence of SEQ ID NO: 183.

In a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186 and in that the second polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 174 and SEQ ID NO: 175 and SEQ ID NO: 183.

In one aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 176 and in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 177.

In a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 176 and in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 187.

In yet a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 184 and in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 188 or SEQ ID NO: 189.

In another aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CH1 or CL domain and a second polypeptide containing a CL or CH1 domain, respectively, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domain,

and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other and to the CH1 or CL domain by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof connected by a peptide linker to the CL or CH1 domain of said polypeptide.

In one aspect, provided is a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CH1 domain and a second polypeptide containing a CL domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domain, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the CH1 domain by a peptide linker and in that the second polypeptide comprises one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to the CL domain of said polypeptide.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CL domain and a second polypeptide containing a CH1 domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domain, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the CL domain by a peptide linker and in that the second polypeptide comprises one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to the CH1 domain of said polypeptide.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to TnC is an antibody fragment.

In particular, the moiety capable of specific binding to TnC is an antigen binding moiety.

In particular, the moiety capable of specific binding to TnC is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, and aVH and a scaffold antigen binding protein.

In particular, the TNF family ligand trimer-containing antigen binding molecule comprises one or two moieties capable of specific binding to TnC.

In a particular aspect, the invention is concerned with a TNF family ligand trimer-containing antigen binding molecule as defined above, wherein the moiety capable of specific binding to TnC is a Fab molecule.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to TnC antigen is a scaffold antigen binding protein.

The invention provides a TNF family ligand trimer-containing antigen binding molecule that comprises at least one moiety capable of specific binding to TnC. In a particular aspect, the TNF family ligand trimer-containing antigen binding molecule comprises one moiety capable of specific binding to TnC. In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising two moieties capable of specific binding TnC.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the moiety capable of specific binding to TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 71, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 60.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the moiety capable of specific binding to TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57.

In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the moiety capable of specific binding to TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence SEQ ID NO: 71, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 59, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 60.

In one aspect, provided is a TNF family ligand trimer-containing antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to TnC comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 46 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 45 or wherein the moiety capable of specific binding to TnC comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 48 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 47.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule according to the invention, wherein a peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to the CH1 or CL domain of a heavy chain by a second peptide linker and wherein one ectodomain of said TNF ligand family member or a fragment thereof is fused at the its C-terminus the CL or CH1 domain on a light chain by a third peptide linker.

In a particular aspect, the invention relates to a TNF family ligand trimer-containing antigen binding molecule as defined above, wherein the peptide linker is (G4S)₂, i.e. a peptide linker of SEQ ID NO: 150. In one aspect, the first peptide linker is (G4S)₂, the second peptide linker is GSPGSSSSGS (SEQ ID NO: 153) and the third peptide linker is (G4S)₂. In another aspect, the first, the second and the third peptide linker is (G4S)₂.

The invention is further concerned with a TNF family ligand trimer-containing antigen binding molecule as defined herein before, comprising an Fc domain composed of a first and a second subunit capable of stable association.

In particular, the TNF family ligand trimer-containing antigen binding molecule of the invention comprising (c) an Fc domain composed of a first and a second subunit capable of stable association further comprises (a) a Fab molecule capable of specific binding to a target cell antigen, wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of a CH2 domain in the Fc domain.

In a further aspect, the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is an IgG1 Fc domain. In a particular aspect, the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain.

In another aspect, the invention is concerned with a TNF family ligand trimer-containing antigen binding molecule as defined herein before, comprising

(c) an Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular towards Fcγ receptor.

In particular, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of the IgG heavy chains. More particularly, provided is a trimeric TNF family ligand-containing antigen binding molecule according to the invention which comprises an IgG1 Fc domain with the amino acid substitutions L234A, L235A and P329G (EU numbering).

In a further aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC, a first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker fused at its C-terminus by a second peptide linker to a second heavy or light chain, and a second peptide comprising one ectodomain of said TNF ligand family member fused at its C-terminus by a third peptide linker to a second light or heavy chain, respectively.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a CH1 domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a CL domain that is part of a light chain.

In yet another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a CL domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a CH1 domain that is part of a light chain.

In a further aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a VH domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a VL domain that is part of a light chain.

Provided is further a TNF family ligand trimer-containing antigen binding molecule, wherein in the CL domain adjacent to the TNF ligand family member the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been substituted by lysine (K), and wherein in the CH1 domain adjacent to the TNF ligand family member the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) have been substituted by glutamic acid (E).

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein before, wherein the antigen binding molecule comprises

(a) a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC,

(b) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186, and a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183.

In one aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

(i) a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 45 or a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 48 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 47, (ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 178, and (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 179.

In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

(i) a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 45 or a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 48 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 47, (ii) a second heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 108, SEQ ID NO: 116 and SEQ ID NO: 120, and (iii) a second light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 109, SEQ ID NO: 117 and SEQ ID NO: 121.

In yet another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain and the second polypeptide contains a CH3 domain, respectively, and wherein the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the C-terminus of the CH3 domain by a peptide linker and wherein the second polypeptide comprises one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to C-terminus of the CH3 domain of said polypeptide.

In a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain and wherein the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 176 and SEQ ID NO: 184 and the second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172 and SEQ ID NO: 183. In one aspect, the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof comprises an amino acid sequence SEQ ID NO: 176 and the second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 172. In a particular aspect, the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof comprises an amino acid sequence of SEQ ID NO: 184 and the second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 183.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) one Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain. The invention thus relates to a TNF family ligand trimer-containing antigen binding molecule, wherein TNF family ligand trimer-containing antigen binding molecule is monovalent for the binding to the target cell antigen.

In a further aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule as defined before further comprising (d) a Fab domain that is not capable of specific binding to TnC. Thus, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association,

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain, and (d) a Fab domain that is not capable of specific binding to a target cell antigen.

In particular, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein before comprising

(i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 127, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 125, and one light chain comprising the amino acid sequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO:130, a second heavy chain comprising the amino acid sequence of SEQ ID NO:131, and one light chain comprising the amino acid sequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 124, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 133, and one light chain comprising the amino acid sequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 136, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 137, and one light chain comprising the amino acid sequence of SEQ ID NO: 77.

In particular, such a TNF family ligand trimer-containing antigen binding molecule comprises two moieties capable of specific binding to TnC. In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising two moieties capable of specific binding to TnC. In particular, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein before comprising

(i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 112, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 113, and two light chains comprising the amino acid sequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 124, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 125, and two light chains comprising the amino acid sequence of SEQ ID NO: 77.

In one aspect, the TNF ligand family member is OX40L. In a further aspect, the ectodomain of a TNF ligand family member comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 181 or SEQ ID NO: 182, particularly the amino acid sequence SEQ ID NO: 181.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein before, comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 190 or SEQ ID: 191 and in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 181 or SEQ ID NO: 182.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule as describe herein, wherein the target cell antigen is Tenascin-C (TnC) and the moiety capable of specific binding to TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 71, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 60.

Particularly, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein, wherein the antigen binding molecule comprises

(i) a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO:45 or a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 48 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 47, (ii) a second heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 195, and (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 196.

According to another aspect of the invention, there is provided an isolated polynucleotide encoding an antigen binding molecule as defined herein before. The invention further provides a vector, particularly an expression vector, comprising the isolated polynucleotide of the invention and a host cell comprising the isolated polynucleotide or the vector of the invention. In some embodiments the host cell is a eukaryotic cell, particularly a mammalian cell.

In another aspect, provided is a method for producing the antigen binding molecule of the invention, comprising the steps of (i) culturing the host cell of the invention under conditions suitable for expression of the antigen binding molecule, and (ii) recovering the antigen binding molecule. The invention also encompasses an antigen binding molecule produced by the method of the invention.

The invention further provides a pharmaceutical composition comprising the antigen binding molecule of the invention and at least one pharmaceutically acceptable excipient.

Also encompassed by the invention is the antigen binding molecule of the invention, or the pharmaceutical composition of the invention, for use as a medicament. In one aspect is provided the antigen binding molecule of the invention, or the pharmaceutical composition of the invention, for use in the treatment of a disease in an individual in need thereof. In a specific embodiment, provided is the antigen binding molecule of the invention, or the pharmaceutical composition of the invention, for use in the treatment of cancer.

Also provided is the use of the antigen binding molecule of the invention for the manufacture of a medicament for the treatment of a disease in an individual in need thereof, in particular for the manufacture of a medicament for the treatment of cancer, as well as a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the antigen binding molecule of the invention in a pharmaceutically acceptable form. In a specific embodiment, the disease is cancer. In any of the above embodiments the individual is preferably a mammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C shows immunohistological staining in LS174T xenografts tumors at 100× magnification as stained with anti-TnC clone 18D4 (FIG. 1B) and anti-TnC clone 11C7 (FIG. 1C). The pattern of staining corresponds to specific TnC stroma fibers. The TnC staining, for both clones 18D4 and 11C7, is overall expressed with moderate intensity. Negative isotype control signal validates the specificity of the technique (FIG. 1A).

FIG. 2 shows the results of the immunohistological staining in human tumor array with a rabbit isotype control. Negative isotype control signal in all tissues tested validates the specificity of the technique.

FIG. 3 shows the results of the immunohistological staining in human tumor array with anti-TnC clone 18D4. The pattern of staining corresponds to specific TnC stroma fibers. The TnC staining is expressed at higher levels in most tumor tissues compared to control normal pair tissue.

FIG. 4 shows the results of the immunohistological staining in human tumor array with anti-TnC clone 11C7. The pattern of staining corresponds to specific TnC stroma fibers. The TnC staining is expressed at higher levels in most tumor tissues compared to control normal pair tissue.

FIGS. 5A and 5B show components for the assembly of monovalent TnC targeted split trimeric human 4-1BB ligand (71-254) (construct 6.1). FIG. 5A shows a dimeric ligand fused to human IgG1-CL domain. FIG. 5B shows a monomeric ligand fused to human IgG1-CH1 domain. “GGGGSGGGGS” is disclosed as SEQ ID NO: 150.

FIG. 6 shows the 4-1BBL-trimer-containing antigen binding molecule constructs 6.1 of the invention (monovalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih) PGLALA fusion containing CH-CL cross with charged residues). The preparation and production of this construct is described in Example 1.1. The VH and VL domains are those of anti-TnC antibody 18D4, the thick black point stands for the knob-into-hole modification. * symbolizes amino acid modifications in the CH1 and CL domain (so-called charged residues).

FIGS. 7A and 7B show the components for the assembly of monovalent TnC targeted split trimeric human 4-1BB ligand (71-254) (construct 6.2). FIG. 7A shows dimeric ligand fused to human IgG1-CL domain. FIG. 7B shows monomeric ligand fused to human IgG1-CH1 domain. “GGGGSGGGGS” is disclosed as SEQ ID NO: 150.

FIG. 8 shows monovalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion containing CH-CL cross without charged residues (construct 6.2). The preparation and production of this construct is described in Example 1.2. The VH and VL domains are those of anti-TnC antibody 18D4, the thick black point stands for the knob-into-hole modification.

FIGS. 9A and 9B show components for the assembly of bivalent TnC targeted split trimeric human 4-1BB ligand (71-254) (construct 6.3). FIG. 9A shows dimeric ligand fused to human IgG1 Fc hole chain. FIG. 9B shows monomeric ligand fused to human IgG1 Fc knob chain. “GGGGSGGGGS” is disclosed as SEQ ID NO: 150.

FIG. 10 shows bivalent TnC (18D4) targeted split trimeric 4-1BB ligand Fc (kih) PGLALA fusion (construct 6.3). The preparation and production of this construct is described in Example 1.3. The VH and VL domains are those of anti-TnC antibody 18D4, the thick black point stands for the knob-into-hole modification.

FIGS. 11A and 11B show components for the assembly of monovalent TnC targeted split trimeric human 4-1BB ligand (71-248) (construct 6.4). FIG. 11A shows dimeric ligand fused to human IgG1-CL domain. FIG. 11B shows monomeric ligand fused to human IgG1-CH1 domain. “GGGGSGGGGS” is disclosed as SEQ ID NO: 150.

FIG. 12 monovalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross with charged residues (construct 6.4). The preparation and production of this construct is described in Example 1.4. The VH and VL domains are those of anti-TnC antibody 18D4, the thick black point stands for the knob-into-hole modification.

FIGS. 13A and 13B show components for the assembly of monovalent TnC targeted split trimeric human 4-1BB ligand (71-248) (construct 6.5). FIG. 13A shows dimeric ligand fused to human IgG1-CL domain. FIG. 13B shows monomeric ligand fused to human IgG1-CH1 domain. “GGGGSGGGGS” is disclosed as SEQ ID NO: 150.

FIG. 14 shows monovalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross without charged residues (construct 6.5). The preparation and production of this construct is described in Example 1.5. The VH and VL domains are those of anti-TnC antibody 18D4, the thick black point stands for the knob-into-hole modification.

FIGS. 15A and 15B show components for the assembly of bivalent TnC targeted split trimeric human 4-1BB ligand (71-248) (construct 6.6). FIG. 15A shows dimeric ligand fused to human IgG1 Fc hole chain. FIG. 15B shows monomeric ligand fused to human IgG1 Fc knob chain. “GGGGSGGGGS” is disclosed as SEQ ID NO: 150.

FIG. 16 shows bivalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.6). The preparation and production of this construct is described in Example 1.6. The VH and VL domains are those of anti-TnC antibody 18D4, the thick black point stands for the knob-into-hole modification.

FIGS. 17A and 17B show components for the assembly of monovalent targeted split trimeric human 4-1BB ligand fused at the C-terminus of a human Fc. FIG. 17A shows dimeric ligand fused to human IgG1 Fc hole or knob chain. FIG. 17B shows monomeric ligand fused to human IgG1 Fc knob or hole chain. “GGGGSGGGGS” is disclosed as SEQ ID NO: 150.

FIGS. 18A and 18B show monovalent targeted split trimeric C-terminal 4-1BB ligand Fc (kih) knob fusion. FIG. 18A shows construct 6.11, dimeric 4-1BBL fused to hole Fc chain and monomeric 4-1BBL fused to knob Fc chain. FIG. 18B shows construct 6.12, dimeric 4-1BBL fused to knob Fc chain and monomeric 4-1BBL fused to hole Fc chain. The preparation and production of these constructs is described in Example 1.7. The VH and VL domains are those of anti-TnC antibody 18D4, the thick black point stands for the knob-into-hole modification.

FIGS. 19A and 19B show monovalent targeted (hole chain) split trimeric C-terminal 4-1BB ligand Fc (kih) fusion. FIG. 19A shows construct 6.13, dimeric 4-1BBL fused to hole Fc chain and monomeric 4-1BBL fused to knob Fc chain. FIG. 19B shows construct 6.14, dimeric 4-1BBL fused to knob Fc chain and monomeric 4-1BBL fused to hole Fc chain. The preparation and production of these constructs is described in Example 1.8. The VH and VL domains are those of anti-TnC antibody 18D4, the thick black point stands for the knob-into-hole modification.

FIGS. 20A, 20B, and 20C relate to simultaneous binding of targeted split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion molecules. FIG. 20A shows setup of the SPR experiments. FIG. 20B shows simultaneous binding of human 4-1BB and human TnC for construct 6.5. FIG. 20C shows simultaneous binding of human 4-1BB and human TnC for construct 6.6.

FIGS. 21A, 21B, 21C, and 21D show binding of TnC-targeted 4-1BB split trimeric ligand Fc fusion antigen binding molecules to freshly isolated PBMCs. Shown is the geo mean of fluorescence intensity of PE-conjugated secondary detection antibody (y-axis) versus the concentration of tested constructs (x-axis). Binding was monitored on fresh human CD45+CD3+ CD8neg CD4+ T cells (FIG. 21A), fresh human CD45+ CD3+ CD4neg CD8+ T cells (FIG. 21), fresh human CD45+ CD3neg CD19+ B cells (FIG. 21C) and CD45+ CD3+ CD4neg CD8neg γδT cells (FIG. 21D).

FIGS. 22A, 22B, 22C, and 22D show binding of TnC-targeted 4-1BB split trimeric ligand Fc fusion antigen binding molecules to activated PBMCs. Shown is the geo mean of fluorescence intensity of PE-conjugated secondary detection antibody (y-axis) versus the concentration of tested constructs (x-axis). Binding was monitored on activated human CD45+ CD3+ CD8neg CD4+ T cells (FIG. 22A), activated human CD45+ CD3+ CD4neg CD8+ T cells (FIG. 22B), CD45+ CD3neg CD19+ B cells (FIG. 22C) and activated human CD45+ CD3+ CD4neg CD8neg γδT cells (FIG. 22D).

FIG. 23 shows binding of TnC-targeted or untargeted split trimeric human 4-1BB ligand Fc (kih) fusion molecules to human-TnC expressing U87-MG tumor cell line. Binding was detected with R-Phycoerythrin-fluorochrome conjugated anti-human IgG Fcγ-specific goat IgG F(ab′)2 fragment and the geo mean measured by flow cytometry is shown on the y-axis. On the x-axis the used concentration of TnC-targeted 4-1BB split trimeric ligand Fc fusion antigen binding molecules and their controls are shown.

FIG. 24 shows an illustration of an activation assay set up with human 4-1BB expressing HeLa reporter cell line. A crosslinking of 4-1BB, expressed on the reporter cells, induces NFκB activation and NFκB-mediated Luciferase expression. After lysis of the cells, luciferase can catalyze the oxidation of Luciferin to Oxyluciferin. This chemical reaction correlates positively with the strength of NFκB-mediated luciferase expression and can be measured by the strength of light emission (units of released light).

FIGS. 25A and 25B show the NFκB-activation-induced luciferase activity. Units of released light (URL) are measured for 0.5 s/well and plotted against the used concentration of split trimeric 4-1BB ligand containing fusion molecules and controls. Human 4-1BB-expressing HeLa-reporter cells were incubated for 6 h in the absence (FIG. 25A) or presence of crosslinking via human-TnC expressing U87MG cells (FIG. 25B). The cell ratio of 4-1BB-expressing HeLa reporter cell to TnC-expressing tumor cells was 1:5.

FIG. 26 shows an illustration of the Schematic set-up of the human PBMC activation in the presence of TnC-expressing U87MG, agonistic anti-human CD3 antibody and bispecific anti-TnC split trimeric 4-1BBL fusion molecule construct 6.5.

FIGS. 27A and 27B shows the human PBMC activation assay in the presence of TnC+U87MG, agonistic CD3 antibody and TnC-targeted split trimeric 4-1BBL fusion molecules. FIG. 27A shows expression levels of CD25 as frequency of total CD8+ T cells are plotted against the used concentration of construct 6.5 and 6.6 or controls (control F, control L and control E). FIG. 27B shows levels of CD137 (4-1BB) as frequency of total CD8+ T cells are plotted against the used concentration of construct 6.5 and 6.6 or controls (control F, control L and control E).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as generally used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.

As used herein, the term “antigen binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant Examples of antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins.

As used herein, the term “moiety capable of specific binding” refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one aspect, the antigen binding moiety is able to activate signaling through its target cell antigen. In a particular aspect, the antigen binding moiety is able to direct the entity to which it is attached (e.g., the TNF family ligand trimer) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant Moieties capable of specific binding to a target cell antigen include antibodies and fragments thereof as further defined herein. In addition, moieties capable of specific binding to a target cell antigen include scaffold antigen binding proteins as further defined herein, e.g., binding domains which are based on designed repeat proteins or designed repeat domains (see e.g., WO 2002/020565).

In relation to an antibody or fragment thereof, the term “moiety capable of specific binding” refers to the part of the molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. A moiety capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Particularly, a moiety capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. The term “bispecific” means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

The term “valent” as used within the current application denotes the presence of a specified number of binding sites in an antigen binding molecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen binding molecule.

The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g., Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g., U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. As used herein, thus, the term “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteins from the antibody hinge region. Fab′-SH are Fab′ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab′)₂ fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.

The term “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e., the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). This crossover Fab molecule is also referred to as CrossFab_((VLVH)). On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is also referred to as CrossFab_((CLCH)).

A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CH1 domain. In addition, these single chain Fab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

A “crossover single chain Fab fragment” or “x-scFab” is a is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL form together an antigen-binding site which binds specifically to an antigen and wherein said linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (V_(H)) and light chains (V_(L)) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V_(H) with the C-terminus of the V_(L), or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, e.g. described in Houston, J. S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.

“Scaffold antigen binding proteins” are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In one aspect of the invention, a scaffold antigen binding protein is selected from the group consisting of CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-domain (Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrin repeat protein (DARPin), a variable domain of antibody light chain or heavy chain (single-domain antibody, sdAb), a variable domain of antibody heavy chain (nanobody, aVH), V_(NAR) fragments, a fibronectin (AdNectin), a C-type lectin domain (Tetranectin); a variable domain of a new antigen receptor beta-lactamase (V_(NAR) fragments), a human gamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domain of human protease inhibitors, microbodies such as the proteins from the knottin family, peptide aptamers and fibronectin (adnectin).

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptor expressed on mainly CD4+ T-cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to CDRs of antibodies can be substituted with heterologous sequence to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies (e.g. U.S. Pat. No. 7,166,697B1). Evibodies are around the same size as the isolated variable region of an antibody (e.g., a domain antibody). For further details see Journal of Immunological Methods 248 (1-2), 31-45 (2001).

Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 and US20070224633.

An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen. The domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP 1641818A1.

Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulfide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

A transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the Trans-body. For further details see J. Biol. Chem 274, 24066-24073 (1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33 residue motif consisting of two alpha-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.

A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single domain antibodies were derived from the variable domain of the antibody heavy chain from camelids (nanobodies or V_(H)H fragments). Furthermore, the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or V_(NAR) fragments derived from sharks.

Fibronectin is a scaffold which can be engineered to bind to antigen. Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the .beta.-sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site. For further details see Expert Opin. Biol. Ther. 5, 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges—examples of microproteins include KalataBl and conotoxin and knottins. The microproteins have a loop which can be engineered to include up to 25 amino acids without affecting the overall fold of the microprotein. For further details of engineered knottin domains, see WO2008098796.

An “antigen binding molecule that binds to the same epitope” as a reference molecule refers to an antigen binding molecule that blocks binding of the reference molecule to its antigen in a competition assay by 50% or more, and conversely, the reference molecule blocks binding of the antigen binding molecule to its antigen in a competition assay by 50% or more.

The term “antigen binding domain” refers to the part of an antigen binding molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope”, and refers to a site (e.g., a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). The proteins useful as antigens herein can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. In a particular embodiment the antigen is a human protein. Where reference is made to a specific protein herein, the term encompasses the “full-length”, unprocessed protein as well as any form of the protein that results from processing in the cell. The term also encompasses naturally occurring variants of the protein, e.g., splice variants or allelic variants.

The terms “capable of specific binding to TnC” refer to an antigen binding molecule or antigen binding moiety that is capable of binding Tenascin-C (TnC) with sufficient affinity such that the antigen binding molecule is useful as a diagnostic and/or therapeutic agent in targeting TnC. The antigen binding molecules include but are not limited to, antibodies, Fab molecules, crossover Fab molecules, single chain Fab molecules, Fv molecules, scFv molecules, single domain antibodies, and VH and scaffold antigen binding protein. In one embodiment, the extent of binding of an anti-TnC antigen binding molecule to an unrelated, non-TnC protein is less than about 10% of the binding of the antigen binding molecule to TnC as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antigen binding molecule that binds to TnC has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤5 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹M to 10⁻¹³ M, e.g., from 10 nM to 0.1 nM, e.g., from 5 nM to 0.1 nM, e.g., from 2 nM to 0.1 nM). In certain embodiments, an anti-TnC antigen binding molecule binds to an epitope of TnC that is conserved among TnC from different species. In certain embodiments, an antigen binding molecule that binds to an epitope of TnC is specific for at least one of the domains selected from the group consisting of A1, A2, A3, A4, B, AD1, AD2, C and D. In certain embodiments an antigen binding molecule specific for the TnC A1 and TnC A4 domains is provided. In certain embodiments an antigen binding molecule specific for the TnC C domain is provided. By “specific binding” is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding molecule to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the binding of the antigen binding molecule to the antigen as measured, e.g., by SPR. In certain embodiments, a molecule that binds to the antigen has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤5 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g. from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (k_(off) and k_(on), respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

A “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell or in the environment of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma. In certain embodiments, the target cell antigen is expressed in the extracellular matrix in or around tumor tissue. In particular, the target cell antigen is Tenascin-C (TnC). The term “Tenascin-C” or “TnC” as used herein, refers to any native TnC from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkey) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TnC as well as any form of TnC that results from processing in the cell. The term also encompasses naturally occurring variants of TnC, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human TnC antigen sequence (with N-terminal GST and 6×His-tag; and C-terminal avi-tag and 6×His-tag) is shown in SEQ ID NO: 4. The amino acid sequence of an exemplary mouse TnC antigen sequence (with N-terminal GST and 6×His-tag; and C-terminal avi-tag and 6×His-tag) is shown in SEQ ID NO: 5. The amino acid sequence of an exemplary cynomolgus TnC antigen sequence (with N-terminal GST and 6×His-tag (SEQ ID NO: 221); and C-terminal avi-tag and 6×His-tag) is shown in SEQ ID NO: 6. In the human TnC molecule, up to nine alternatively spliced fibronectin-type III domains, which may be inserted between the fifth and the sixth of the constant fibronectin-type III domains are known (for a schematic representation of the domain structure of TnC, see e.g., Orend and Chiquet-Ehrismann, Cancer Letters 244, 143-163 (2006). Similarly, in the mouse TnC molecule, six alternatively spliced fibronectin-type III domains are described (e.g., in Joestner and Faissner, J Biol Chem 274, 17144-17151 (1999)).

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

The term “hypervariable region” or “HVR,” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).) Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-32 26-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102 V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR3 89-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A is according to the numbering conventions set forth by Kabat et al. (see below). ²“AbM” with a lowercase “b” as used in Table A refers to the CDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

As used herein, the term “affinity matured” in the context of antigen binding molecules (e.g., antibodies) refers to an antigen binding molecule that is derived from a parent antigen binding molecule, e.g., by mutation, binds to the same antigen, preferably binds to the same epitope, as the parent antibody; and has a higher affinity for the antigen than that of the reference antigen binding molecule. Affinity maturation generally involves modification of one or more amino acid residues in one or more CDRs of the antigen binding molecule. Typically, the affinity matured antigen binding molecule binds to the same epitope as the initial parent antigen binding molecule.

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ respectively.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of “humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding.

A “human” antibody is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The “CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e., from an amino acid residue at about position 341 to an amino acid residue at about position 447 of an IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced “protuberance” (“knob”) in one chain thereof and a corresponding introduced “cavity” (“hole”) in the other chain thereof; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as herein described. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

The “knob-into-hole” (kih) technology is described e.g., in U.S. Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). The numbering is according to EU index of Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

A “region equivalent to the Fc region of an immunoglobulin” is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)). The term “effector functions” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

An “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt accession no. P08637, version 141).

The term “TNF ligand family member” or “TNF family ligand” refers to a proinflammatory cytokine. Cytokines in general, and in particular the members of the TNF ligand family, play a crucial role in the stimulation and coordination of the immune system. At present, nineteen cytokines have been identified as members of the TNF (tumor necrosis factor) ligand superfamily on the basis of sequence, functional, and structural similarities. All these ligands are type II transmembrane proteins with a C-terminal extracellular domain (ectodomain), N-terminal intracellular domain and a single transmembrane domain. The C-terminal extracellular domain, known as TNF homology domain (THD), has 20-30% amino acid identity between the superfamily members and is responsible for binding to the receptor. The TNF ectodomain is also responsible for the TNF ligands to form trimeric complexes that are recognized by their specific receptors.

Members of the TNF ligand family are selected from the group consisting of Lymphotoxin α (also known as LTA or TNFSF1), TNF (also known as TNFSF2), LTβ (also known as TNFSF3), OX40L (also known as TNFSF4), CD40L (also known as CD154 or TNFSF5), FasL (also known as CD95L, CD178 or TNFSF6), CD27L (also known as CD70 or TNFSF7), CD30L (also known as CD153 or TNFSF8), 4-1BBL (also known as TNFSF9), TRAIL (also known as APO2L, CD253 or TNFSF10), RANKL (also known as CD254 or TNFSF11), TWEAK (also known as TNFSF12), APRIL (also known as CD256 or TNFSF13), BAFF (also known as CD257 or TNFSF13B), LIGHT (also known as CD258 or TNFSF14), TL1A (also known as VEGI or TNFSF15), GITRL (also known as TNFSF18), EDA-A1 (also known as ectodysplasin A1) and EDA-A2 (also known as ectodysplasin A2). The term refers to any native TNF family ligand from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. In specific embodiments of the invention, the TNF ligand family member is selected from the group consisting of OX40L, FasL, CD27L, TRAIL, 4-1BBL, CD40L and GITRL. In a particular embodiment, the TNF ligand family member is selected from 4-1BBL and OX40L.

Further information, in particular sequences, of the TNF ligand family members may be obtained from publically accessible databases such as Uniprot (www.uniprot.org). For instance, the human TNF ligands have the following amino acid sequences: human Lymphotoxin α (UniProt accession no. P01374, SEQ ID NO: 203), human TNF (UniProt accession no. P01375, SEQ ID NO: 204), human Lymphotoxin β (UniProt accession no. Q06643, SEQ ID NO: 205), human OX40L (UniProt accession no. P23510, SEQ ID NO: 206), human CD40L (UniProt accession no. P29965, SEQ ID NO: 207), human FasL (UniProt accession no. P48023, SEQ ID NO: 208), human CD27L (UniProt accession no. P32970, SEQ ID NO: 209), human CD30L (UniProt accession no. P32971, SEQ ID NO: 210), 4-1BBL (UniProt accession no. P41273, SEQ ID NO: 211), TRAIL (UniProt accession no. P50591, SEQ ID NO: 212), RANKL (UniProt accession no. 014788, SEQ ID NO: 213), TWEAK (UniProt accession no. 043508, SEQ ID NO: 214), APRIL (UniProt accession no. 075888, SEQ ID NO: 215), BAH- (UniProt accession no. Q9Y275, SEQ ID NO: 216), LIGHT (UniProt accession no. 043557, SEQ ID NO: 217), TL1A (UniProt accession no. 095150, SEQ ID NO: 218), GITRL (UniProt accession no. Q9UNG2, SEQ ID NO: 219) and ectodysplasin A (UniProt accession no. Q92838, SEQ ID NO: 220).

An “ectodomain” is the domain of a membrane protein that extends into the extracellular space (i.e., the space outside the target cell). Ectodomains are usually the parts of proteins that initiate contact with surfaces, which leads to signal transduction. The ectodomain of TNF ligand family member as defined herein thus refers to the part of the TNF ligand protein that extends into the extracellular space (the extracellular domain), but also includes shorter parts or fragments thereof that are responsible for the trimerization and for the binding to the corresponding TNF receptor. The term “ectodomain of a TNF ligand family member or a fragment thereof” thus refers to the extracellular domain of the TNF ligand family member that forms the extracellular domain or to parts thereof that are still able to bind to the receptor (receptor binding domain).

The term “costimulatory TNF ligand family member” or “costimulatory TNF family ligand” refers to a subgroup of TNF ligand family members, which are able to costimulate proliferation and cytokine production of T-cells. These TNF family ligands can costimulate TCR signals upon interaction with their corresponding TNF receptors and the interaction with their receptors leads to recruitment of TNFR-associated factors (TRAF), which initiate signalling cascades that result in T-cell activation. Costimulatory TNF family ligands are selected from the group consisting of 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, more particularly the costimulatory TNF ligand family member is selected from 4-1BBL and OX40L.

As described herein before, 4-1BBL is a type II transmembrane protein and one member of the TNF ligand family Complete or full length 4-1BBL having the amino acid sequence of SEQ ID NO: 212 has been described to form trimers on the surface of cells. The formation of trimers is enabled by specific motives of the ectodomain of 4-1BBL. Said motives are designated herein as “trimerization region”. The amino acids 50-254 of the human 4-1BBL sequence (SEQ ID NO: 180) form the extracellular domain of 4-1BBL, but even fragments thereof are able to form the trimers. In specific embodiments of the invention, the term “ectodomain of 4-1BBL or a fragment thereof” refers to a polypeptide having an amino acid sequence selected from SEQ ID NO: 175 (amino acids 52-254 of human 4-1BBL), SEQ ID NO: 172 (amino acids 71-254 of human 4-1BBL), SEQ ID NO: 174 (amino acids 80-254 of human 4-1BBL) and SEQ ID NO: 173 (amino acids 85-254 of human 4-1BBL) or a polypeptide having an amino acid sequence selected from SEQ ID NO: 183 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 194 (amino acids 52-248 of human 4-1BBL), SEQ ID NO: 193 (amino acids 80-248 of human 4-1BBL) and SEQ ID NO: 192 (amino acids 85-248 of human 4-1BBL), but also other fragments of the ectodomain capable of trimerization are included herein.

As described herein before, OX40L is another type II transmembrane protein and a further member of the TNF ligand family Complete or full length human OX40L has the amino acid sequence of SEQ ID NO: 206. The amino acids 51-183 of the human OX40L sequence (SEQ ID NO: 181) form the extracellular domain of OX40L, but even fragments thereof that are able to form the trimers. In specific embodiments of the invention, the term “ectodomain of OX40L or a fragment thereof” refers to a polypeptide having an amino acid sequence selected from SEQ ID NO: 181 (amino acids 51-183 of human OX40L) or SEQ ID NO: 182 (amino acids 52-183 of human OX40L), but also other fragments of the ectodomain capable of trimerization are included herein.

The term “peptide linker” refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides are, for example, (G₄S)_(n) (SEQ ID NO: 222), (SG₄)_(n) (SEQ ID NO: 223) or G₄(SG₄)_(n) (SEQ ID NO: 224) peptide linkers, wherein “n” is generally a number between 1 and 10, typically between 1 and 4, in particular 2, i.e., the peptides selected from the group consisting of GGGGS (SEQ ID NO: 162), GGGGSGGGGS (SEQ ID NO: 150), SGGGGSGGGG (SEQ ID NO: 151) and GGGGSGGGGSGGGG (SEQ ID NO: 152), but also include the sequences GSPGSSSSGS (SEQ ID NO: 153), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 154), GSGSGNGS (SEQ ID NO: 155), GGSGSGSG (SEQ ID NO: 156), GGSGSG (SEQ ID NO: 157), GGSG (SEQ ID NO: 158), GGSGNGSG (SEQ ID NO: 159), GGNGSGSG (SEQ ID NO: 160) and GGNGSG (SEQ ID NO: 161). Peptide linkers of particular interest are (G4S)₁ (SEQ ID NO: 162) or GGGGS (SEQ ID NO: 162), (G₄S)₂ (SEQ ID NO: 150) or GGGGSGGGGS (SEQ ID NO: 150) and GSPGSSSSGS (SEQ ID NO: 153), more particularly (G₄S)₂ (SEQ ID NO: 150) or GGGGSGGGGS (SEQ ID NO: 150) and GSPGSSSSGS (SEQ ID NO: 153).

The term “amino acid” as used within this application denotes the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

A “single chain fusion protein” as used herein refers to a single chain polypeptide composed of one or two ectodomains of said TNF ligand family member fused to a part of antigen binding moiety or Fc part. The fusion may occur by directly linking the N or C-terminal amino acid of the antigen binding moiety via a peptide linker to the C- or N-terminal amino acid of the ectodomain of said TNF ligand family member.

By “fused” or “connected” is meant that the components (e.g., a polypeptide and an ectodomain of said TNF ligand family member) are linked by peptide bonds, either directly or via one or more peptide linkers.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN. SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

In certain embodiments, “amino acid sequence variants” of the TNF ligand trimer-containing antigen binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the TNF ligand trimer-containing antigen binding molecules Amino acid sequence variants of the TNF ligand trimer-containing antigen binding molecules may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecules, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. Sites of interest for substitutional mutagenesis include the HVRs and Framework (FRs). Conservative substitutions are provided in Table B under the heading “Preferred Substitutions” and further described below in reference to amino acid side chain classes (1) to (6) Amino acid substitutions may be introduced into the molecule of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE B Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;     -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;     -   (3) acidic: Asp, Glu;     -   (4) basic: His, Lys, Arg;     -   (5) residues that influence chain orientation: Gly, Pro;     -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

The term “amino acid sequence variants” includes substantial variants wherein there are amino acid substitutions in one or more hypervariable region residues of a parent antigen binding molecule (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antigen binding molecule and/or will have substantially retained certain biological properties of the parent antigen binding molecule. An exemplary substitutional variant is an affinity matured antibody, which may be generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antigen binding molecules displayed on phage and screened for a particular biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antigen binding molecule to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antigen binding molecule complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include TNF family ligand trimer-containing antigen binding molecule with an N-terminal methionyl residue. Other insertional variants of the molecule include the fusion of the N- or C-terminus to a polypeptide which increases the serum half-life of the TNF ligand trimer-containing antigen binding molecules.

In certain embodiments, the TNF family ligand trimer-containing antigen binding molecules provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation variants of the molecules may be obtained by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the TNF ligand trimer-containing antigen binding molecule comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in TNF family ligand trimer-containing antigen binding molecule may be made in order to create variants with certain improved properties. In one aspect, variants of TNF family ligand trimer-containing antigen binding molecules are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. Such fucosylation variants may have improved ADCC function, see e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Further variants of the TNF family ligand trimer-containing antigen binding molecules of the invention include those with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and/or improved ADCC function, see for example WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function and are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, it may be desirable to create cysteine engineered variants of the TNF family ligand trimer-containing antigen binding molecule of the invention, e.g., “thioMAbs,” in which one or more residues of the molecule are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the molecule. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antigen binding molecules may be generated as described, e.g., in U.S. Pat. No. 7,521,541.

In certain aspects, the TNF family ligand trimer-containing antigen binding molecules provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc. In another aspect, conjugates of an antibody and non-proteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety are killed.

In another aspect, immunoconjugates of the TNF family ligand trimer-containing antigen binding molecules provided herein may be obtained. An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

“Cross-species reactivity” refers to the ability of certain antibodies to specifically bind to their respective target antigen wherein said target antigen may derive from different species (e.g., human, mouse, cynomolgus, etc.). A cross-species reactive antibody binds to its respective target antigen derived from at least two different species with a K_(D) value lower than about 1 μM, preferably lower than about 100 nM, more preferably lower than about 10 nM, more preferably lower than about 5 nM, most preferably lower than about 2 nM. The term “binds to its respective target antigen derived from at least two different species with a K_(D) value lower than” means that the respective antibody binds to the target antigen deriving from each of the indicated species with a dissociation constant K_(D) lower than the indicated K_(D) value. In preferred embodiments a cross-species reactive antibody binds to the target antigen from all indicated species with similar affinity, preferably within a K_(D) range of 10 nM to 0.1 nM, more preferably 5 nM to 0.1 nM, most preferably 2 nM to 0.1 nM. In some embodiment, similar affinity for the antigen derived from several species, which means binding of the target antigen within a narrow K_(D) range (e.g., within a range of 10 nM to 0.1 nM or narrower) for all species of interest, is advantageous, e.g., for diagnostic assays or animal models of human diseases. In further preferred embodiment, a cross-species reactive antibody binds to the target antigen from all indicated species (e.g., human, mouse and cynomolgus monkey) with similar affinity, in particular within a K_(D) range of a factor of 100, within a K_(D) range of a factor of 50, within a K_(D) range of a factor of 20, within a K_(D) range of a factor of 10, within a K_(D) range of a factor of 5. In a preferred embodiment, a cross-species reactive antibody binds to the target antigen from human, mouse and cynomolgus monkey with similar affinity, in particular within a K_(D) range of a factor of 10. For clarity, the cross-species reactive antibody binds to one of the indicated species with highest affinity compared to the other indicated species. Accordingly, the cross-species reactive antibody binds to one of the indicated species with lowest affinity compared to the other indicated species. Within a K_(D) range of a defined factor X means that the affinity for the indicated species with hightest affinity is not more than X-times higher than the affinity for the indicated species with lowest affinity. In other words, the K_(D) value for the indicated species with lowest affinity is not more than X-times the K_(D) value for the indicated species with highest affinity. It is clear to the field that any method for measuring affinity or avidity can be used to verify that a cross-species reactive antibody binds to the target antigen from all indicated species within a given K_(D) factor range as described herein as long as the same conditions are applied to the K_(D) measurement for all indicated species. Preferably, the K_(D) values are measured using SPR, in particular at 25° C. Preferably, the affinities are measured using the cross-species reactive antibody as Fab fragment.

The term “polynucleotide” refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g., ALIGN-2).

The term “expression cassette” refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expression construct” and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, e.g., mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER. C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.

An “effective amount” of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.

A “therapeutically effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Particularly, the individual or subject is a human.

The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable excipient” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable excipient includes, but is not limited to, a buffer, a stabilizer, or a preservative.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the molecules of the invention are used to delay development of a disease or to slow the progression of a disease.

The term “cancer” as used herein refers to proliferative diseases, such as lymphomas, carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colorectal cancer (CRC), pancreatic cancer, breast cancer, triple-negative breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, melanoma, multiple myeloma, B-cell cancer (lymphoma), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

Compositions and Methods

Distinct alternatively spliced isoforms of Tenascin-C (TnC), are specifically expressed in certain pathological conditions but essentially absent from healthy adult tissues, thus antigen binding molecules targeting TnC have great therapeutic potential. The present invention provides novel TNF family ligand trimer-containing antigen binding molecules with particularly advantageous properties such as producibility, stability, binding affinity, biological activity, targeting efficiency and reduced toxicity. The invention further provides antigen binding molecules that bind to TnC, in particular antigen binding molecules with improved affinity and cross-species reactivity. Antigen binding molecules of the invention are useful, e.g., for the diagnosis or treatment of diseases characterized by expression of TnC, such as cancer.

In a one aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the target cell antigen is Tenascin-C (TnC). In a further aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to Tenascin-C (TnC) and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof.

In a particular aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof, and (c) an Fc domain composed of a first and a second subunit capable of stable association.

In a particular aspect, the TNF family ligand trimer-containing antigen binding molecule comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof, wherein the TNF ligand family member costimulates human T-cell activation.

In another particular aspect, the TNF family ligand trimer-containing antigen binding molecule comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof, wherein the ectodomains of a TNF ligand family member are identical in all instances.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein, comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that

-   -   (i) the first polypeptide contains a CH1 or CL domain and the         second polypeptide contains a CL or CH1 domain, respectively,         wherein the second polypeptide is linked to the first         polypeptide by a disulfide bond between the CH1 and CL domain,         and wherein the first polypeptide comprises two ectodomains of a         TNF ligand family member or fragments thereof that are connected         to each other and to the CH1 or CL domain by a peptide linker         and wherein the second polypeptide comprises one ectodomain of         said TNF ligand family member or a fragment thereof connected         via a peptide linker to the CL or CH1 domain of said         polypeptide, or     -   (ii) the first polypeptide contains a CH3 domain and the second         polypeptide contains a CH3 domain, respectively, and wherein the         first polypeptide comprises two ectodomains of a TNF ligand         family member or fragments thereof that are connected to each         other and to the C-terminus of the CH3 domain by a peptide         linker and wherein the second polypeptide comprises only one         ectodomain of said TNF ligand family member or a fragment         thereof connected via a peptide linker to C-terminus of the CH3         domain of said polypeptide, or     -   (iii) the first polypeptide contains a VH-CL or a VL-CH1 domain         and the second polypeptide contains a VL-CH1 domain or a VH-CL         domain, respectively, wherein the second polypeptide is linked         to the first polypeptide by a disulfide bond between the CH1 and         CL domain, and wherein the first polypeptide comprises two         ectodomains of a TNF ligand family member or fragments thereof         that are connected to each other and to VH or VL by a peptide         linker and wherein the second polypeptide comprises one         ectodomain of said TNF ligand family member or a fragment         thereof connected via a peptide linker to VL or VH of said         polypeptide.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule of claim 1, comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that

-   -   (i) the first polypeptide contains a CH1 or CL domain and the         second polypeptide contains a CL or CH1 domain, respectively,         wherein the second polypeptide is linked to the first         polypeptide by a disulfide bond between the CH1 and CL domain,         and wherein the first polypeptide comprises two ectodomains of a         TNF ligand family member or fragments thereof that are connected         to each other and to the CH1 or CL domain by a peptide linker         and wherein the second polypeptide comprises one ectodomain of         said TNF ligand family member or a fragment thereof connected         via a peptide linker to the CL or CH1 domain of said         polypeptide, or     -   (ii) the first polypeptide contains a CH3 domain and the second         polypeptide contains a CH3 domain, respectively, and wherein the         first polypeptide comprises two ectodomains of a TNF ligand         family member or fragments thereof that are connected to each         other and to the C-terminus of the CH3 domain by a peptide         linker and wherein the second polypeptide comprises only one         ectodomain of said TNF ligand family member or a fragment         thereof connected via a peptide linker to C-terminus of the CH3         domain of said polypeptide.

In a particular aspect, the TNF family ligand trimer-containing antigen binding molecule comprises a TNF ligand family member that costimulates human T-cell activation which is selected from 4-1BBL and OX40L. More particularly, the TNF ligand family member is 4-1BBL.

In another aspect, wherein the ectodomain of a TNF ligand family member comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 183, SEQ ID NO: 192, SEQ ID NO: 193 and SEQ ID NO: 194, particularly the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 183. In one aspect, the ectodomain of a TNF ligand family member or fragment thereof comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183, particularly the amino acid sequence of SEQ ID NO: 172 or SEQ ID NO: 183. In a particular aspect, the ectodomain of a TNF ligand family member or fragment thereof comprises the amino acid sequence of SEQ ID NO: 183.

In a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186 and in that the second polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183. In a particular aspect, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 184 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 183.

In one aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 176 and in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 177.

In a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 176 and in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 187.

In yet a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 184 and in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 188 or SEQ ID NO: 189.

In another aspect, the TNF ligand family member is OX40L. In a particular aspect, provided is TNF family ligand trimer-containing antigen binding molecule, wherein the ectodomain of a TNF ligand family member comprises the amino acid sequence of SEQ ID NO: 181 or SEQ ID NO: 182, particularly the amino acid sequence of SEQ ID NO: 181.

In one aspect, the invention relates to a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence of SEQ ID NO: 190 or SEQ ID: 191 and in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 181 or SEQ ID NO: 182, respectively.

In one aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CH1 or CL domain and a second polypeptide containing a CL or CH1 domain, respectively, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domain, and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the CH1 or CL domain by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to the CL or CH1 domain of said polypeptide.

In one aspect, provided is a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CH1 domain and a second polypeptide containing a CL domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domain,

and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the CH1 domain by a peptide linker and in that the second polypeptide comprises one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to the CL domain of said polypeptide.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CL domain and a second polypeptide containing a CH1 domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domain,

and wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the CL domain by a peptide linker and in that the second polypeptide comprises one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to the CH1 domain of said polypeptide.

In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof.

In yet another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) more than one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to said polypeptide.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) two moities capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof.

In a particular aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain and the second polypeptide contains a CH3 domain, respectively, and wherein the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the C-terminus of the CH3 domain by a peptide linker and wherein the second polypeptide comprises one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to C-terminus of the CH3 domain of said polypeptide. Particularly, such TNF family ligand trimer-containing antigen binding molecule comprises two moieties capable of specific binding to TnC.

In a further aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule as defined herein before, wherein the moiety capable of specific binding to TnC is selected from the group consisting of an antibody, an antibody fragment and a scaffold antigen binding protein.

In one aspect, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein before, wherein the moiety capable of specific binding to TnC is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, an aVH and a scaffold antigen binding protein. In one aspect, the moiety capable of specific binding to TnC is an aVH or a scaffold antigen binding protein. In one aspect, the moiety capable of specific binding to a target cell antigen is a scaffold antigen binding protein capable of specific binding to a target cell antigen.

In particular, the TNF family ligand trimer-containing antigen binding molecule comprises one or two moieties capable of specific binding to TnC.

In a particular aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the moiety capable of specific binding to TnC is a Fab molecule or a crossover Fab molecule capable of specific binding to a target cell antigen. In particular, the moiety capable of specific binding to TnC is a Fab molecule.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule according to the invention, wherein the antigen binding molecule comprises a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC, a first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker fused at its C-terminus by a second peptide linker to a second heavy or light chain, and a second peptide comprising one ectodomain of said TNF ligand family member fused at its C-terminus by a third peptide linker to a second light or heavy chain, respectively.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule according to the invention, wherein a peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to the CH1 domain of a heavy chain by a second peptide linker and wherein one ectodomain of said TNF ligand family member or a fragment thereof is fused at the its C-terminus to the CL domain on a light chain by a third peptide linker.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule according to the invention, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a CH1 domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a CL domain that is part of a light chain.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule according to the invention, wherein a peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to the CL domain of a heavy chain by a second peptide linker and wherein one ectodomain of said TNF ligand family member or a fragment thereof is fused at the its C-terminus to the CH1 domain on a light chain by a third peptide linker.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule according to the invention, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a CL domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a CH1 domain that is part of a light chain.

In a further aspect, the invention is concerned with a TNF family ligand trimer-containing antigen binding molecule according to the invention, wherein a peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus to the CL domain of a light chain by a second peptide linker and wherein one ectodomain of said TNF ligand family member or a fragment thereof is fused at the its C-terminus to the CH1 domain of the heavy chain by a third peptide linker.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule according to the invention, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a VH domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a VL domain that is part of a light chain.

In a particular aspect, the invention relates to a TNF family ligand trimer-containing antigen binding molecule as defined above, wherein the peptide linker is (G4S)₂ (SEQ ID NO: 150). In one aspect, the first peptide linker is (G4S)₂ (SEQ ID NO: 150), the second peptide linker is GSPGSSSSGS (SEQ ID NO: 153) and the third peptide linker is (G4S)₂ (SEQ ID NO: 150). In particular, the invention relates to a TNF ligand trimer-containing antigen binding molecule as defined above, wherein the first peptide linker is (G4S)₂ (SEQ ID NO: 150), the second peptide linker is (G4S)₂ (SEQ ID NO: 150), and the third peptide linker is (G4S)₂ (SEQ ID NO: 150).

In another aspect, the TNF family ligand trimer-containing antigen binding molecule as defined herein before comprises an Fc domain composed of a first and a second subunit capable of stable association.

In particular, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises (a) a Fab molecule capable of specific binding to TnC, wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of a CH2 domain in the Fc domain and (c) an Fc domain composed of a first and a second subunit capable of stable association.

In a further aspect, the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is an IgG1 Fc domain. In a particular aspect, the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain.

Fc Domain Modifications Reducing Fc Receptor Binding and/or Effector Function

The Fc domain of the TNF family ligand trimer-containing antigen binding molecules of the invention consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other.

The Fc domain confers favorable pharmacokinetic properties to the antigen binding molecules of the invention, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the bispecific antibodies of the invention to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Accordingly, in particular aspects, the Fc domain of the TNF family ligand trimer-containing antigen binding molecule of the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG1 Fc domain. In one aspect, the Fc does not substantially bind to an Fc receptor and/or does not induce effector function. In a particular aspect the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc domain does not induce effector function. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

In certain aspects, one or more amino acid modifications may be introduced into the Fc region of a antigen binding molecule provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In a particular aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof, and (c) an Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular towards Fcγ receptor.

In one aspect, the Fc domain of the antigen binding molecule of the invention comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In particular, the Fc domain comprises an amino acid substitution at a position of E233, L234, L235, N297, P331 and P329 (EU numbering). In particular, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of the IgG heavy chains. More particularly, provided is an antigen binding molecule according to the invention which comprises an Fc domain with the amino acid substitutions L234A, L235A and P329G (“PGLALA”, EU numbering) in the IgG heavy chains. The amino acid substitutions L234A and L235A refer to the so-called LALA mutation. The “PGLALA” combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of a human IgG1 Fc domain and is described in International Patent Appl. Publ. No. WO 2012/130831 A1 which also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions. “EU numbering” refers to the numbering according to EU index of Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

In another aspect, the Fc domain is an IgG4 Fc domain. IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgG1 antibodies. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position 5228 (Kabat numbering), particularly the amino acid substitution S228P. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising amino acid substitutions L235E and S228P and P329G (EU numbering). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are also described in WO 2012/130831.

Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g., by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. A suitable such binding assay is described herein. Alternatively, binding affinity of Fc domains or cell activating bispecific antigen binding molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing FcγIIIa receptor.

Effector function of an Fc domain, or antigen binding molecule of the invention comprising an Fc domain, can be measured by methods known in the art. A suitable assay for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component, specifically to C1q, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. C1q binding assays may be carried out to determine whether the antigen binding molecule of the invention is able to bind C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

In a particular aspect, the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain.

Fc Domain Modifications Promoting Heterodimerization

In one aspect, the TNF family ligand trimer-containing antigen binding molecules of the invention comprise

(a) at least one moiety capable of specific binding to TnC,

(b) a first and a second polypeptide that are linked to each other by a disulfide bond,

wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof, and (c) an Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular to Fcγ receptor. Thus, they comprise different moieties, fused to one or the other of the two subunits of the Fc domain that are typically comprised in two non-identical polypetide chains (“heavy chains”). Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the TNF family ligand trimer-containing antigen binding molecules in recombinant production, it will thus be advantageous to introduce in the Fc domain of the TNF family ligand trimer-containing antigen binding molecules of the invention a modification promoting the association of the desired polypeptides.

Accordingly, the Fc domain of the TNF family ligand trimer-containing antigen binding molecules of the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, said modification is particularly in the CH3 domain of the Fc domain

In a specific aspect, said modification is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain. Thus, in a particular aspect, the invention relates to a TNF family ligand trimer-containing antigen binding molecule as described herein before which comprises an IgG molecule, wherein the Fc part of the first heavy chain comprises a first dimerization module and the Fc part of the second heavy chain comprises a second dimerization module allowing a heterodimerization of the two heavy chains of the IgG molecule and the first dimerization module comprises knobs and the second dimerization module comprises holes according to the knob into hole technology.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).

Accordingly, in a particular aspect, in the CH3 domain of the first subunit of the Fc domain of the TNF family ligand trimer-containing antigen binding molecules of the invention an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.

In a specific aspect, in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). More particularly, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A). More particularly, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain. The disulfide bridge further stabilizes the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In an alternative aspect, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g., as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.

Modifications in the CH1/CL Domains

To further improve correct pairing, the TNF family ligand trimer-containing antigen binding molecules can contain different charged amino acid substitutions (so-called “charged residues”). These modifications are introduced in the crossed or non-crossed CH1 and CL domains. In a particular aspect, the invention relates to a TNF family ligand trimer-containing antigen binding molecule, wherein in one of CL domains the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been substituted by lysine (K) and wherein in one of the CH1 domains the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) have been substituted by glutamic acid (E).

More particularly, the invention relates to a TNF family ligand trimer-containing antigen binding molecule, wherein in the CL domain adjacent to the TNF ligand family member the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been substituted by lysine (K), and wherein in the CH1 domain adjacent to the TNF ligand family member the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) have been substituted by glutamic acid (E).

Particular TNF Family Ligand Trimer-Containing Antigen Binding Molecules

In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC, a first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker fused at its C-terminus by a second peptide linker to a second heavy or light chain, and a second peptide comprising one ectodomain of said TNF ligand family member fused at its C-terminus by a third peptide linker to a second light or heavy chain, respectively.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a CH1 domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a CL domain that is part of a light chain.

In yet another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a CL domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof that is fused at its C-terminus by a third peptide linker to a CH1 domain that is part of a light chain.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a VH domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a VL domain that is part of a light chain.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule as described herein, wherein in the CL domain adjacent to the TNF ligand family member the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been substituted by lysine (K), and wherein in the CH1 domain adjacent to the TNF ligand family member the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) have been substituted by glutamic acid (E). These modifications lead to so-called charged residues with advantageous properties that avoid undesired effects such as for example mispairing.

In one aspect, provided is a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to the second subunit of the Fc domain comprising the knob mutations and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the first subunit of the Fc domain comprising the hole mutations.

In a particular aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to the second subunit of the Fc domain comprising the knob mutations and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the first subunit of the Fc domain comprising the hole mutations and wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of the second subunit of the Fc domain comprising the knob mutations.

In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to the second subunit of the Fc domain comprising the knob mutations and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the first subunit of the Fc domain comprising the hole mutations and wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of the first subunit of the Fc domain comprising the hole mutations.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to the first subunit of the Fc domain comprising the hole mutations and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the second subunit of the Fc domain comprising the knob mutations.

In a particular aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to the first subunit of the Fc domain comprising the hole mutations and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the second subunit of the Fc domain comprising the knob mutations and wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of the second subunit of the Fc domain comprising the knob mutations.

In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to the first subunit of the Fc domain comprising the hole mutations and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the second subunit of the Fc domain comprising the knob mutations and wherein the Fab heavy chain is fused at the C-terminus to the N-terminus of the first subunit of the Fc domain comprising the hole mutations.

Particular TnC Binding Moieties

In one aspect, the present invention provides for TnC binding moieties. In another aspect, TNC binding moieties of the present invention can be included in bivalent or multivalent binding molecules as described herein. In one embodiment the moiety capable of specific binding to TnC is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, and aVH and a scaffold antigen binding protein. In another aspect, the present invention provides for antigen binding molecules comprising one or more moiety capable of specific binding to TnC. The molecules of the invention comprising a TnC binding moiety as described herein have a high affinity for one ore more TnC domains and/or cross-species reactivity.

In one embodiment, an anti-TnC antigen binding molecule of the invention comprises at least one (e.g., one, two, three, four, five, or six) heavy or light chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, or a variant or truncated form thereof containing at least the specificity-determining residues (SDRs) for said CDR.

In one embodiment, an antigen binding molecule of the invention comprises at least one, at least two, or all three heavy chain CDR (HCDR) sequences selected from (a) HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 67 and SEQ ID NO: 70; (b) HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 68 and SEQ ID NO: 71; and (c) HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 69 and SEQ ID NO: 72. In a further embodiment, the antigen binding molecule comprises a heavy chain variable region comprising (a) a heavy chain CDR1 selected from the group consisting of SEQ ID NO: 67 and SEQ ID NO: 70; (b) a heavy chain CDR2 selected from the group consisting of SEQ ID NO: 68 and SEQ ID NO: 71; and (c) a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 69 and SEQ ID NO: 72, or variants or truncated forms thereof containing at least the SDRs for said CDRs.

In one embodiment, an antigen binding molecule of the invention comprises at least one, at least two, or all three light chain CDR (LCDR) sequences selected from (a) LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55 and SEQ ID NO: 58; (b) LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 56 and SEQ ID NO: 59; and (c) LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 60. In a further embodiment, the antigen binding molecule comprises a light chain variable region comprising (a) a light chain CDR1 selected from the group consisting of SEQ ID NO: 55 and SEQ ID NO: 58 (b) a light chain CDR2 selected from the group consisting of SEQ ID NO: 56 and SEQ ID NO: 59; and (c) a light chain CDR3 selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 60, or variants or truncated forms thereof containing at least the SDRs for said CDRs.

In one embodiment, an antigen binding molecule of the invention comprises a heavy chain variable region comprising a heavy chain CDR1 selected from the group consisting of SEQ ID NO: 67 and SEQ ID NO: 70; a heavy chain CDR2 selected from the group consisting of SEQ ID NO: 68 and SEQ ID NO: 71; and a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 69 and SEQ ID NO: 72, and a light chain variable region comprising a light chain CDR1 selected from the group consisting of SEQ ID NO: 55 and SEQ ID NO: 58; a light chain CDR2 selected from the group consisting of SEQ ID NO: 56 and SEQ ID NO: 59; and a light chain CDR3 selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 60, or variants or truncated forms thereof containing at least the SDRs for said CDRs.

In yet another specific embodiment, an antigen binding molecule of the invention comprises a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 67; the heavy chain CDR2 of SEQ ID NO: 68; and the heavy chain CDR3 of SEQ ID NO: 69, and a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 55; the light chain CDR2 of SEQ ID NO: 56; and the light chain CDR3 of SEQ ID NO: 57.

In yet another specific embodiment, an antigen binding molecule of the invention comprises a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 70; the heavy chain CDR2 of SEQ ID NO: 71; and the heavy chain CDR3 of SEQ ID NO: 72, and a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 58, the light chain CDR2 of SEQ ID NO: 59; and the light chain CDR3 of SEQ ID NO: 60.

In one embodiment, an antigen binding molecule of the invention comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48. In one embodiment, the antigen binding molecule comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 46 and SEQ ID NO: 48.

In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TnC antigen binding molecule comprising that sequence retains the ability to bind to TnC. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 46 or SEQ ID NO: 48. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs or CDRs (i.e., in the FRs). Optionally, an anti-TnC antigen binding molecule according to the invention comprises the VH sequence in SEQ ID NO: 46 or SEQ ID NO: 48, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three heavy chain CDRs selected from the sequences set forth in SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72 for the HCDR1, HCDR2 and HCDR3.

In another embodiment, an antigen binding molecule of the invention comprises a light chain variable region comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a sequence selected from the group consisting of SEQ ID NO: 45 and SEQ ID NO: 47. In yet another embodiment, the antigen binding molecule comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 45 and SEQ ID NO: 47.

In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TnC antigen binding molecule comprising that sequence retains the ability to bind to TnC. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 45 or SEQ ID NO: 47. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs or CDRs (i.e., in the FRs). Optionally, an anti-TnC antigen binding molecule of the invention comprises the VL sequence in SEQ ID NO: 45 or SEQ ID NO: 47, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three light chain CDRs selected from sequences set forth in SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60 for the LCDR1, LCDR2 and LCDR3.

In another aspect, an anti-TnC antigen binding molecule is provided, wherein the antigen binding molecule comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antigen binding molecule comprises a heavy chain variable region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of: SEQ ID NO: 46 and SEQ ID NO: 48, and a light chain variable region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of: SEQ ID NO: 45 and SEQ ID NO: 47. In one embodiment, the antigen binding molecule comprises the VH and VL sequences in SEQ ID NO: 46 or SEQ ID NO: 48 and SEQ ID NO: 45 or SEQ ID NO: 47, respectively, including post-translational modifications of those sequences.

In a specific embodiment, an antigen binding molecule of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 45. In a specific embodiment, an antigen binding molecule of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47. In a particular embodiment, the antigen binding molecule according to any of the above embodiments additionally comprises an Fc region or a region equivalent to the Fc region of an immunoglobulin. In one embodiment an antigen binding molecule of the invention comprises an Fc region, particularly a IgG Fc region, most particularly a IgG1 Fc region. In a particular embodiment, the antigen binding molecule of the invention is a full length antibody, particularly an IgG class antibody, most particularly an IgG1 isotype antibody. In another embodiment, the TnC binding moiety of the antigen binding molecule of the invention is an antibody fragment, selected from the group of: an scFv fragment, an Fv fragment, a Fab fragment, and a F(ab′)2 fragment. In a further embodiment, the antigen binding molecule of the invention is an antibody fragment having an Fc region, or a fusion protein that comprises a region equivalent to the Fc region of an immunoglobulin. In one embodiment, the antigen binding molecule of the invention is a monoclonal antibody. In one embodiment, an antigen binding molecule of the invention is chimeric, more specifically humanized. In a particular embodiment, an antigen binding molecule of the invention is human. In another embodiment, an antigen binding molecule of the invention comprises a human constant region. In one embodiment the antigen binding molecule of the invention comprises a human Fc region, preferably a human IgG Fc region, most particularly a human IgG1 Fc region.

In one embodiment, an antigen binding molecule of the invention comprises a heavy chain constant region, wherein said heavy chain constant region is a human IgG constant region, particularly a human IgG1 constant region, comprising an Fc region. In one embodiment, an antigen binding molecule of the invention comprises a heavy chain region, wherein said heavy chain region is a human IgG heavy chain region, particularly a human IgG1 heavy chain region, comprising an Fc region. In a specific embodiment, the antigen binding molecule comprises a heavy chain region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 83, and SEQ ID NO: 84. In another specific embodiment an antigen binding molecule of the invention comprises a light chain region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 77 and SEQ ID NO: 79. In yet another specific embodiment, an antigen binding molecule of the invention comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 78, and a light chain region comprising the amino acid sequence of SEQ ID NO: 77. In yet another specific embodiment, an antigen binding molecule of the invention comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 80, and a light chain region comprising the amino acid sequence of SEQ ID NO: 79.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to TnC, wherein said antigen binding molecule comprises a) a heavy chain region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 78 and SEQ ID NO: 80, or a light chain region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 77 and SEQ ID NO: 79, or a combination thereof, comprising an Fc region or a region equivalent to the Fc region of an immunoglobulin.

In one embodiment, an antigen binding molecule of the invention binds to Tenascin-C (TnC) with a dissociation constant (K_(D)) value lower than about 1 μM to about 0.001 nM, particularly a K_(D) value lower than about 100 nM, lower than about 10 nM, or lower than about 1 nM. In a specific embodiment, an antigen binding molecule of the invention binds to human Tenascin-C (TnC) with a dissociation constant (K_(D)) value lower than about 1 nM. In one embodiment, an antigen binding molecule of the invention binds to human, mouse, and cynomolgus TnC. In one embodiment, an antigen binding molecule of the invention has cross-species reactivity. In another specific embodiment, an antigen binding molecule of the invention binds to the C domain of human, mouse, and cynomolgus TnC. In one embodiment the antigen binding molecule of the invention has cross-species reactivity. In one embodiment the antigen binding molecule of the present invention binds to at least one of human, mouse and cynomolgus TnC with a K_(D) value lower than about 100 nM, lower than 10 nM, lower than 5 nM or lower than 2 nM. In a specific embodiment the antigen binding molecule of the present invention binds to at least one of human, mouse and cynomolgus TnC with a K_(D) value lower than about 2 nM. In further embodiments, the antigen binding molecule of the present invention binds to human TnC with a first K_(D) value K_(D)1, wherein said antigen binding molecule binds to mouse TnC with a second K_(D) value K_(D)2, and wherein said antigen binding molecule binds to cynomolgus TnC with a third K_(D) value K_(D)3, wherein all of the K_(D) values selected from the group consisting of K_(D)1, K_(D)2 and K_(D)3 are lower than about 10 nM, lower than about 5 nM or lower than about 2 nM. In yet further embodiments, the antigen binding molecule of the present invention binds to human TnC with a first K_(D) value K_(D)1, wherein said antigen binding molecule binds to mouse TnC with a second K_(D) value K_(D)2, and wherein said antigen binding molecule binds to cynomolgus TnC with a third K_(D) value K_(D)3, wherein all of the K_(D) values selected from the group consisting of K_(D)1, K_(D)2 and K_(D)3 are in the range of 10 nM to 0.1 nM, in the range of 5 nM to 0.1 nM or in the range of 2 nM to 0.1 nM. In a further embodiment, the antibody of the present invention binds to human TnC with a first K_(D) value K_(D)1, wherein said antibody binds to mouse TnC with a second K_(D) value K_(D)2, and wherein said antibody binds to cynomolgus TnC with a third K_(D) value K_(D)3, wherein all of the K_(D) values selected from the group consisting of K_(D)1, K_(D)2 and K_(D)3 are within a K_(D) range of a factor of 20. In a further embodiment, the antibody of the present invention binds to human TnC with a first K_(D) value K_(D)1, wherein said antibody binds to mouse TnC with a second K_(D) value K_(D)2, and wherein said antibody binds to cynomolgus TnC with a third K_(D) value K_(D)3, wherein all of the K_(D) values selected from the group consisting of K_(D)1, K_(D)2 and K_(D)3 are within a K_(D) range of a factor of 10. In one embodiment an antigen binding molecule of the invention is specific for at least one of the TnC domain selected from the group consisting of A1, A2, A3, A4, B, AD1, AD2, C and D. In one embodiment, an antigen binding molecule is provided, wherein said antigen binding molecule is able to bind to at least one of the TnC domain selected from the group consisting of A1, A4 and C. In one embodiment, an antigen binding molecule of the invention is specific for the TnC domains A1 and A4. In one embodiment, an antigen binding molecule of the invention is specific for the TnC domain C. In a specific embodiment, an antigen binding molecule of the invention binds to the A1 and to the A4 domain of human, mouse, and cynomolgus TnC. In another specific embodiment, an antigen binding molecule of the invention binds to the C domain of human, mouse, and cynomolgus TnC. In one embodiment the antigen binding molecule of the invention has cross-species reactivity. In a more specific embodiment, an antigen binding molecule of the invention binds to the A1 domain of human, mouse and cynomolgus TnC A1 and to the A4 domain of human, mouse and cynomolgus TnC A4 with a K_(D) value lower than about 100 nM, lower than about 10 nM, lower than about 5 nM or lower than about 2 nM. K_(D) values are determined by Surface Plasmon Resonance, using the antibodies as Fab or IgG. In one embodiment, an anti-TnC antigen binding molecule of the invention binds TnC in human tissues.

In one embodiment, an antigen binding molecule of the invention comprises an Fc region, wherein said antigen binding molecule comprises at least one amino acid substitution in the Fc region. In one embodiment, an antigen binding molecule of the invention comprising at least one amino acid substitution in the Fc region has decreased effector function and/or decreased Fc receptor binding affinity compared to an antigen binding molecule comprising the parent non-substituted Fc region. In a specific embodiment said parent non-substituted Fc region comprises the amino acid residues Leu234, Leu235 and Pro329, wherein the substituted Fc region comprises at least one of the amino acid substitutions selected from the group consisting of Leu234Ala, Leu235Ala and Pro329Gly relative to the parent non-substituted Fc region. In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to TnC, wherein said antigen binding molecule comprises a) a heavy chain region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 83 and SEQ ID NO: 84, and a light chain region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 77 and SEQ ID NO: 79. In a specific embodiment, antibodies of the invention comprise a heavy chain region comprising an amino acid sequence selected from the group of: SEQ ID NO: 83, and SEQ ID NO: 84. In a further embodiment antigen binding molecule comprising the substituted Fc region has decreased effector function and/or decreased Fc receptor binding affinity compared to the antigen binding molecule comprising the parent non-substituted heavy chain region. In a further specific embodiment, the antigen binding molecule of the invention, comprising said substituted Fc region, comprises the amino acid substitutions Leu234Ala, Leu235Ala and Pro329Gly relative to the parent non-substituted Fc region, wherein binding to FcγR and C1q is abolished and/or wherein Fc-mediated effector function is abolished. In a particular embodiment, the decreased effector function is decreased ADCC. In another particular embodiment, the decreased effector function is abolished ADCC. The decreased Fc receptor binding preferably is decreased binding to an activating Fc receptor, most preferably FcγRIIIa. In one embodiment, an antigen binding molecule of the invention does not cause a clinically significant level of toxicity when administered to an individual in a therapeutically effective amount.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to TnC, wherein said antigen binding molecule comprises a) a heavy chain variable region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48, or a light chain variable region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 45 and SEQ ID NO: 47, or a combination thereof, and b) an Fc region or a region equivalent to the Fc region of an immunoglobulin. In one embodiment, an antigen binding molecule of the invention comprises an Fc region, wherein said Fc region is a glycoengineered Fc region. In a further embodiment, an antigen binding molecule of the invention is glycoengineered to have modified oligosaccharides in the Fc region. In a specific embodiment, the antigen binding molecule has an increased proportion of bisected oligosaccharides in the Fc region, compared to a non-glycoengineered antigen binding molecule. In a more specific embodiment, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 50%, more preferably at least about 70%, of the N-linked oligosaccharides in the Fc region of the antigen binding molecule are bisected. The bisected oligosaccharides may be of the hybrid or complex type. In another specific embodiment, an antigen binding molecule of the invention has an increased proportion of non-fucosylated oligosaccharides in the Fc region, compared to a non-glycoengineered antigen binding molecule. In a more specific embodiment, at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 50%, more preferably at least about 70%, of the N-linked oligosaccharides in the Fc region of the antigen binding molecule are non-fucosylated. The non-fucosylated oligosaccharides may be of the hybrid or complex type. In a particular embodiment, an antigen binding molecule of the invention has an increased proportion of bisected, non-fucosylated oligosaccharides in the Fc region, compared to a non-glycoengineered antigen binding molecule. Specifically, the antigen binding molecule comprises an Fc region in which at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 15%, more preferably at least about 25%, at least about 35% or at least about 50%, of the N-linked oligosaccharides are bisected, non-fucosylated. The bisected, non-fucosylated oligosaccharides may be of the hybrid or complex type. In one embodiment, an antigen binding molecule of the invention has increased effector function and/or increased Fc receptor binding affinity. Increased effector function and/or increased Fc receptor binding can result e.g., from glycoengineering and/or affinity maturation of antibodies. In one embodiment, the increased effector function and/or increased Fc receptor binding is a result of glycoengineering of the Fc region of the antigen binding molecule. In another embodiment, the increased effector function and/or increased Fc receptor binding is a result of a combination of increased affinity and glycoengineering. The increased effector function can include, but is not limited to, one or more of the following: increased Fc-mediated cellular cytotoxicity (including increased antibody-dependent cell-mediated cytotoxicity (ADCC)), increased antibody-dependent cellular phagocytosis (ADCP), increased cytokine secretion, increased immune-complex-mediated antigen uptake by antigen-presenting cells, increased binding to NK cells, increased binding to macrophages, increased binding to monocytes, increased binding to polymorphonuclear cells, increased direct signaling inducing apoptosis, increased crosslinking of target-bound antibodies, increased dendritic cell maturation, or increased T cell priming. In a particular embodiment, the increased effector function is increased ADCC. The increased Fc receptor binding preferably is increased binding to an activating Fc receptor, most preferably FcγRIIIa. In one embodiment, an antigen binding molecule of the invention does not cause a clinically significant level of toxicity when administered to an individual in a therapeutically effective amount.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the A1 and A4 domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 46, a light chain variable region comprising the amino acid sequence SEQ ID NO: 45, and a human IgG Fc region.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the C domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 48, a light chain variable region comprising the amino acid sequence SEQ ID NO: 47, and a human IgG Fc region.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the A1 and A4 domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 46, a light chain variable region comprising the amino acid sequence SEQ ID NO: 45, and a human IgG Fc region, and wherein said antigen binding molecule is glycoengineered to have increased effector function and/or Fc receptor binding affinity.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the C domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 48, a light chain variable region comprising the amino acid sequence SEQ ID NO: 47, and a human IgG Fc region, and wherein said antigen binding molecule is glycoengineered to have increased effector function and/or Fc receptor binding affinity.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the A1 and A4 domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 46, a light chain variable region comprising the amino acid sequence SEQ ID NO: 45, and a human IgG Fc region, wherein said antigen binding molecule comprises at least one amino acid substitution in the Fc region, wherein the parent non-substituted Fc region comprises the amino acid residues Leu234, Leu235 and Pro329, wherein the substituted Fc region comprises at least one of the amino acid substitutions selected from the group consisting of Leu234Ala, Leu235Ala and Pro329Gly relative to the parent non-substituted Fc region, wherein the antigen binding molecule comprising the substituted Fc region has decreased effector function and/or decreased Fc receptor binding affinity compared to the antigen binding molecule comprising the parent non-substituted Fc region.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the C domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 48, a light chain variable region comprising the amino acid sequence SEQ ID NO: 47, and a human IgG Fc region, wherein said antigen binding molecule comprises at least one amino acid substitution in the Fc region, wherein the parent non-substituted Fc region comprises the amino acid residues Leu234, Leu235 and Pro329, wherein the substituted Fc region comprises at least one of the amino acid substitutions selected from the group consisting of Leu234Ala, Leu235Ala and Pro329Gly relative to the parent non-substituted Fc region, wherein the antigen binding molecule comprising the substituted Fc region has decreased effector function and/or decreased Fc receptor binding affinity compared to the antigen binding molecule comprising the parent non-substituted Fc region.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the A1 and A4 domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising

(a) the heavy chain CDR1 of SEQ ID NO: 67;

(b) the heavy chain CDR2 of SEQ ID NO: 68;

(c) the heavy chain CDR3 of SEQ ID NO: 69,

and a light chain variable region comprising

(a) the light chain CDR1 of SEQ ID NO: 55;

(b) the light chain CDR2 of SEQ ID NO: 56, and

(c) the light chain CDR3 of SEQ ID NO: 57.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the C domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising

(a) the heavy chain CDR1 of SEQ ID NO: 70;

(b) the heavy chain CDR2 of SEQ ID NO: 71;

(c) the heavy chain CDR3 of SEQ ID NO: 72,

and a light chain variable region comprising

(a) the light chain CDR1 of SEQ ID NO: 58;

(b) the light chain CDR2 of SEQ ID NO: 59, and

(c) the light chain CDR3 of SEQ ID NO: 60.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the A1 and A4 domain of TnC, wherein said antigen binding molecule comprises a heavy chain region comprising the amino acid sequence SEQ ID NO: 78, and a light chain region comprising the amino acid sequence SEQ ID NO: 77.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the A1 and A4 domain of human, mouse and cynomolgus TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 46, a light chain variable region comprising the amino acid sequence SEQ ID NO: 45, and a human IgG Fc region.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the C domain of TnC, wherein said antigen binding molecule comprises a heavy chain region comprising the amino acid sequence SEQ ID NO: 80, and a light chain region comprising an amino acid sequence selected from the group of SEQ ID NO: 79.

In a particular embodiment, the invention provides an antigen binding molecule that specifically binds to the C domain of human, mouse and cynomolgus TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 48, a light chain variable region comprising the amino acid sequence SEQ ID NO: 47, and a human IgG Fc region.

In another particular embodiment, the invention provides an antigen binding molecule that specifically binds to the A1 and A4 domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 46, a light chain variable region comprising the amino acid sequence SEQ ID NO: 45, and a human IgG Fc region, and wherein said antigen binding molecule has an increased proportion of non-fucosylated oligosaccharides and/or an increased proportion of bisected oligosaccharides in said Fc region. In another particular embodiment, the invention provides an antigen binding molecule that specifically binds to the C domain of TnC, wherein said antigen binding molecule comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 48, a light chain variable region comprising the amino acid sequence SEQ ID NO: 47, and a human IgG Fc region, and wherein said antigen binding molecule has an increased proportion of non-fucosylated oligosaccharides and/or an increased proportion of bisected oligosaccharides in said Fc region.

In one aspect, the invention provides for an antigen binding molecule that specifically binds to TnC, wherein said antigen binding molecule comprises at least one amino acid substitution in at least one heavy or light chain CDR of the parent antigen binding molecule. For example, the antigen binding molecule may comprise at least one, e.g., from about one to about ten (i.e., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and particularly from about two to about five, substitutions in one or more hypervariable regions or CDRs (i.e., 1, 2, 3, 4, 5, or 6 hypervariable regions or CDRs) of the parent antigen binding molecule.

Additionally, the antigen binding molecule may also comprise one or more additions, deletions and/or substitutions in one or more framework regions of either the heavy or the light chain, compared to the parent antigen binding molecule. In one embodiment, said at least one amino acid substitution in at least one CDR contributes to increased binding affinity of the antigen binding molecule compared to its parent antigen binding molecule. In another embodiment said antigen binding molecule has at least about 2-fold to about 10-fold greater affinity for TnC than the parent antigen binding molecule (when comparing the antigen binding molecule of the invention and the parent antigen binding molecule in the same format, e.g., the Fab format). Further, the antigen binding molecule derived from a parent antigen binding molecule may incorporate any of the features, singly or in combination, described in the preceding paragraphs in relation to the antibodies of the invention.

TNF Family Ligand Trimer-Containing Antigen Binding Molecules which Particular TnC Binding Moieties

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the moiety capable of specific binding to TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 71, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 60.

In a particular aspect, provided is a TNF family ligand trimer-containing antigen binding molecule of the invention, wherein the moiety capable of specific binding to TnC is a Fab molecule capable of specific binding to TnC and comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule of the invention, wherein the moiety capable of specific binding to TnC is a Fab molecule capable of specific binding to TnC and comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 71 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 59 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 60.

In one aspect, the moiety capable of specific binding to TnC comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 46 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 45 or a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 48 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 47.

In a further aspect, the moiety capable of specific binding to TnC comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45.

In another aspect, the moiety capable of specific binding to TnC comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47.

In a particular aspect, the moiety capable of specific binding to TnC comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 45. In another particular aspect, the moiety capable of specific binding to TnC comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47. In a specific aspect, the moiety capable of specific binding to TnC comprises a VH domain consisting of amino acid sequence of SEQ ID NO: 46 and a VL domain consisting of the amino acid sequence of SEQ ID NO: 45.

In a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC,

(b) a second heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186, and a second light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183.

In a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 45, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186 and in that the second polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183.

In a particular aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding TnC comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 45, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises the the amino acid sequence of SEQ ID NO: 176 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 172.

In a particular aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding TnC comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 45, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises the the amino acid sequence of SEQ ID NO: 184 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 183.

In another aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47, and

a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186 and in that the second polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183.

In a particular aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises the the amino acid sequence of SEQ ID NO: 176 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 172.

In a particular aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding TnC comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises the the amino acid sequence of SEQ ID NO: 184 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 183.

In one aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

(i) a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 45 or

a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 48 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 47,

(ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 178, and

(iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 179.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC, a second heavy chain comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker that is fused at its C-terminus by a second peptide linker to a CH1 domain, and a second light chain comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a CL domain, and wherein the antigen binding molecule comprises

(i) a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 45 or a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 48 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 47, (ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 178, and (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 179.

In a further particular aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC, a second heavy chain comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker and fused at its C-terminus by a second peptide linker to a CL domain, and a second light chain comprising one ectodomain of said TNF ligand family member or a fragment thereof that is fused at its C-terminus by a third peptide linker to a CH1 domain, and wherein the molecule comprises

(i) a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 45 or a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 48 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 47, (ii) a second heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 108, SEQ ID NO: 116 and SEQ ID NO: 120, and (iii) a second light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 109, SEQ ID NO: 117 and SEQ ID NO: 121.

In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

(i) a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 45 or

a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 48 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 47,

(ii) a second heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 102, SEQ ID NO: 108, SEQ ID NO: 116 and SEQ ID NO: 120, and

(iii) a second light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 109, SEQ ID NO: 117 and SEQ ID NO: 121.

In yet another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain and the second polypeptide contains a CH3 domain, respectively, and wherein the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the C-terminus of the CH3 domain by a peptide linker and wherein the second polypeptide comprises one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to C-terminus of the CH3 domain of said polypeptide.

In a further aspect, the TNF family ligand trimer-containing antigen binding molecule of the invention comprises

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain and wherein the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 176 and SEQ ID NO: 184 and the second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172 and SEQ ID NO: 183. In one aspect, the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof comprises an amino acid sequence SEQ ID NO: 176 and the second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 172. In a particular aspect, the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof comprises an amino acid sequence of SEQ ID NO: 184 and the second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 183.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) one Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain. The invention thus relates to a TNF family ligand trimer-containing antigen binding molecule, wherein TNF family ligand trimer-containing antigen binding molecule is monovalent for the binding to the target cell antigen.

In a further aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 46 and a VL domain comprising an amino acid sequence of SEQ ID NO: 45, or a VH domain comprising an amino acid sequence of SEQ ID NO: 48 and a VL domain comprising an amino acid sequence of SEQ ID NO: 47, (b) a Fc domain composed of a first and a second subunit capable of stable association, and (c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain.

In another aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 46 and a VL domain comprising an amino acid sequence of SEQ ID NO: 45, or a VH domain comprising an amino acid sequence of SEQ ID NO: 48 and a VL domain comprising an amino acid sequence of SEQ ID NO: 47, (b) a Fc domain composed of a first and a second subunit capable of stable association, and (c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain and wherein the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 176 and SEQ ID NO: 184 and the second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 172 and SEQ ID NO: 183.

In a particular aspect, provided is a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC comprising a VH domain comprising an amino acid sequence of SEQ ID NO: 46 and a VL domain comprising an amino acid sequence of SEQ ID NO: 45,

(b) a Fc domain composed of a first and a second subunit capable of stable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain and wherein the first polypeptide comprising two ectodomains of a TNF ligand family member comprising an amino acid sequence of SEQ ID NO: 184 and the second polypeptide comprising one ectodomain of said TNF ligand family member comprising the amino acid sequence of SEQ ID NO: 183.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

(i) a light chain comprising the VL domain of the Fab domain capable of specific binding to TnC,

(ii) a fusion polypeptide comprising a first subunit of a Fc domain and a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker, wherein the first polypeptide is fused at its N-terminus to the C-terminus of the first subunit of the Fc domain, and (iii) a heavy chain comprising the VH domain of the Fab domain capable of specific binding to TnC, a second subunit of the Fc domain and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the variable heavy chain of the Fab domain capable of specific binding to TnC is fused at its C-terminus to the N-terminus of the second subunit of the Fc domain and wherein the second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its N-terminus to the C-terminus of the second subunit of the Fc domain.

In a further aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule as defined before further comprising (d) a Fab domain that is not capable of specific binding to TnC. Thus, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable of stable association,

(c) a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the first polypeptide is fused at its N-terminus to the C-terminus to one of the subunits of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the other subunit of the Fc domain, and (d) a Fab domain that is not capable of specific binding to a target cell antigen.

In a further particular aspect of the invention, provided is a TNF family ligand trimer-containing antigen binding molecule comprising

(a) a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC, wherein the first heavy chain comprises the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and the first light chain comprises the VL domain comprising the amino acid sequence of SEQ ID NO: 45, and (b) a second heavy chain comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker that is fused at its C-terminus by a second peptide linker to a CL domain, and a second light chain comprising one ectodomain of said TNF ligand family member or a fragment thereof that is fused at its C-terminus by a third peptide linker to a CH1 domain, wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 120, and the second light chain comprises the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 121. In particular, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 116 and the second light chain comprises the amino acid sequence of SEQ ID NO: 117.

Furthermore, the invention provides a TNF family ligand trimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC, and

(b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide contains a CH3 domain and the second polypeptide contains a CH3 domain, respectively, and wherein the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other and to the C-terminus of the CH3 domain by a peptide linker and wherein the second polypeptide comprises one ectodomain of said TNF ligand family member or a fragment thereof connected via a peptide linker to C-terminus of the CH3 domain of said polypeptide.

In particular, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein before comprising

(i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 127, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 125, and one light chain comprising the amino acid sequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 130, a second heavy chain comprising the amino acid sequence of SEQ ID NO:131, and one light chain comprising the amino acid sequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO:124, a second heavy chain comprising the amino acid sequence of SEQ ID NO:133, and one light chain comprising the amino acid sequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 136, a second heavy chain comprising the amino acid sequence of SEQ ID NO:137, and one light chain comprising the amino acid sequence of SEQ ID NO: 77.

In a particular aspect, such a TNF family ligand trimer-containing antigen binding molecule comprises two moieties capable of specific binding to TnC.

In one aspect, the invention provides a TNF family ligand trimer-containing antigen binding molecule comprising two moieties capable of specific binding to TnC. In particular, provided is a TNF family ligand trimer-containing antigen binding molecule as described herein before comprising

(i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 112, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 113, and two light chains comprising the amino acid sequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 124, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 125, and two light chains comprising the amino acid sequence of SEQ ID NO: 77.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

(i) a light chain comprising the VL domain of the Fab domain capable of specific binding to TNC,

(ii) a fusion polypeptide comprising a first subunit of a Fc domain and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the second polypeptide is fused at its N-terminus to the C-terminus of the first subunit of the Fc domain, and (iii) a heavy chain comprising the VH domain of the Fab domain capable of specific binding to TNC, a second subunit of the Fc domain and a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker, wherein the variable heavy chain of the Fab domain capable of specific binding to TNC is fused at its C-terminus to the N-terminus of the second subunit of the Fc domain and wherein the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof is fused at its N-terminus to the C-terminus of the second subunit of the Fc domain.

In a further aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

(i) a first light chain comprising the VL domain of the Fab domain capable of specific binding to TnC,

(ii) a first heavy chain comprising the VH domain of the Fab domain capable of specific binding to TnC, a first subunit of a Fc domain and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the variable heavy chain of the Fab domain capable of specific binding to TnC is fused at its C-terminus to the N-terminus of the second subunit of the Fc domain and wherein the second polypeptide is fused at its N-terminus to the C-terminus of the first subunit of the Fc domain, (iii) a second heavy chain comprising the VH domain of a Fab domain that is not capable of specific binding to a target cell antigen, a second subunit of the Fc domain and a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker, wherein the variable heavy chain of the Fab domain not capable of specific binding to a target cell antigen is fused at its C-terminus to the N-terminus of the second subunit of the Fc domain and wherein the first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof is fused at its N-terminus to the C-terminus of the second subunit of the Fc domain, and (iv) a second light chain comprising the VL domain of the Fab domain that is not capable of specific binding to a target cell antigen.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

(i) a first light chain comprising the VL domain of the Fab domain capable of specific binding to TnC,

(ii) a first heavy chain comprising the VH domain of the Fab domain capable of specific binding to TnC, a first subunit of a Fc domain and a first polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker, wherein the variable heavy chain of the Fab domain capable of specific binding to TnC is fused at its C-terminus to the N-terminus of the second subunit of the Fc domain and wherein the first polypeptide comprising ectodomains of a TNF ligand family member or fragments thereof is fused at its N-terminus to the C-terminus of the first subunit of the Fc domain, (iii) a second heavy chain comprising the VH domain of a Fab domain that is not capable of specific binding to a target cell antigen, a second subunit of the Fc domain and a second polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof, wherein the variable heavy chain of the Fab domain not capable of specific binding to a target cell antigen is fused at its C-terminus to the N-terminus of the second subunit of the Fc domain and wherein the second polypeptide comprising one ectodomain of a TNF ligand family member or fragments thereof is fused at its N-terminus to the C-terminus of the second subunit of the Fc domain, and (iv) a second light chain comprising the VL domain of the Fab domain that is not capable of specific binding to a target cell antigen.

In further particular aspects, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the antigen binding molecule comprises

a) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 104, a first light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 102 and a second light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 103; or b) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 104, a first light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 108 and a second light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 109; or (c) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 112, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 113 and two light chains comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77; or d) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 104, a first light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 116 and a second light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 117; or e) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 104, a first light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 120 and a second light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 121; or (f) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 124, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 125, and two light chains comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77; or (g) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 127, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 128, and one light chains comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77; or (h) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 130, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 131, and one light chains comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77; or (i) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 124, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 133, and one light chains comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77; or (j) a first heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 136, a second heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 137, and one light chains comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77.

In further particular aspects, the invention relates to a TNF family ligand trimer-containing antigen binding molecule, selected from the group consisting of:

a) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 104, a first light chain comprising the amino acid sequence of SEQ ID NO: 77, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a second light chain comprising the amino acid sequence of SEQ ID NO: 103; b) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 104, a first light chain comprising the amino acid sequence of SEQ ID NO: 77, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 108 and a second light chain comprising the amino acid sequence of SEQ ID NO: 109; (c) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 112, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 113 and two light chains comprising the amino acid sequence of SEQ ID NO: 77; d) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 104, a first light chain comprising the amino acid sequence of SEQ ID NO: 77, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 116 and a second light chain comprising the amino acid sequence of SEQ ID NO: 117; e) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 104, a first light chain comprising the amino acid sequence of SEQ ID NO: 77, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 120 and a second light chain comprising the amino acid sequence of SEQ ID NO: 121; (f) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 124, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 125, and two light chains comprising the amino acid sequence of SEQ ID NO: 77; (g) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 127, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 128, and one light chains comprising the amino acid sequence of SEQ ID NO: 77. (h) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 130, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 131, and one light chains comprising the amino acid sequence of SEQ ID NO: 77. (i) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 124, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 133, and one light chains comprising the amino acid sequence of SEQ ID NO: 77. (j) a molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 136, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 137, and one light chains comprising the amino acid sequence of SEQ ID NO: 77.

In another aspect, provided is a TNF family ligand trimer-containing antigen binding molecule, wherein the TNF ligand family member is OX40L and wherein the target cell antigen is Tenascin-C (TnC) and the moiety capable of specific binding to TnC comprises a VH domain comprising

(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 70,

(ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 71, and

(iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VL domain comprising

(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 58,

(v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and

(vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 60.

In a particular aspect, the TNF family ligand trimer-containing antigen binding molecule of comprises

(i) a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 46 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 45 or a first heavy chain comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 48 and a first light chain comprising the VL domain comprising the amino acid sequence of SEQ ID NO: 47, (ii) a second heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 195, and (iii) a second light chain comprising the amino acid sequence of SEQ ID NO: 196.

Polynucleotides

The invention further provides isolated polynucleotides encoding an antigen binding molecule as described herein or a fragment thereof.

The isolated polynucleotides antigen binding molecules of the invention may be expressed as a single polynucleotide that encodes the entire antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional antigen binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide from the heavy chain portion of the immunoglobulin. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the immunoglobulin.

In some aspects, the isolated polynucleotide encodes the entire antigen binding molecule according to the invention as described herein. In particular, the isolated polynucleotide encodes a polypeptide comprised in the TNF family ligand trimer-containing antigen binding molecule according to the invention as described herein.

In one aspect, the present invention is directed to an isolated polynucleotide encoding a TNF family ligand trimer-containing antigen binding molecule, wherein the polynucleotide comprises (a) a sequence that encodes a moiety capable of specific binding to TnC, (b) a sequence that encodes a polypeptide comprising two ectodomains of a TNF ligand family member or two fragments thereof that are connected to each other by a peptide linker and (c) a sequence that encodes a polypeptide comprising one ectodomain of said TNF ligand family member or a fragment thereof.

In another aspect, provided is an isolated polynucleotide encoding a 4-1BB ligand trimer-containing antigen binding molecule, wherein the polynucleotide comprises (a) a sequence that encodes a moiety capable of specific binding to TnC, (b) a sequence that encodes a polypeptide comprising two ectodomains of 4-1BBL or two fragments thereof that are connected to each other by a peptide linker and (c) a sequence that encodes a polypeptide comprising one ectodomain of 4-1BBL or a fragment thereof.

In one embodiment, the invention is directed to an isolated polynucleotide comprising a sequence encoding one or more (e.g., one, two, three, four, five, or six) of the heavy or light chain complementarity determining regions (CDRs) set forth in SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, or a variant or truncated form thereof containing at least the specificity-determining residues (SDRs) for said CDR.

In another embodiment, the polynucleotide comprises a sequence that encodes three heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3) or three light chain CDRs (e.g., LCDR1, LCDR2, and LCDR3) selected from SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, or variants or truncated forms thereof containing at least the SDRs for each of said three complementarity determining regions. In yet another embodiment, the polynucleotide comprises a sequence encoding three heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3) and three light chain CDRs (e.g., LCDR1, LCDR2, and LCDR3) selected from SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.

In a particular embodiment the polynucleotide encoding one or more CDRs comprises a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of the CDR nucleotide sequences of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.

In a further embodiment, the polynucleotide comprises a sequence encoding a heavy chain variable region selected from the group of SEQ ID NO: 46 and SEQ ID NO: 48, and/or a sequence encoding a light chain variable region selected from the group of SEQ ID NO: 45 and SEQ ID NO: 47. In a particular embodiment, the polynucleotide encoding a heavy chain and/or light chain variable region comprises a sequence selected from the group of variable region nucleotide sequences consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, or a combination thereof.

In a specific embodiment, the polynucleotide comprises a sequence encoding a heavy chain variable region selected from the group of SEQ ID NO: 46 and SEQ ID NO: 48, and a sequence encoding a heavy chain constant region, particularly a human heavy chain constant region. In a particular embodiment, said heavy chain constant region is a human IgG heavy chain constant region, specifically a human IgG1 heavy chain constant region, comprising an Fc region. In another specific embodiment, the polynucleotide comprises a sequence encoding a light chain variable region selected from the group of SEQ ID NO: 45 and SEQ ID NO: 47, and a sequence encoding a light chain constant region, particularly a human light chain constant region.

In one embodiment, the invention is directed to a composition that comprises a first isolated polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 77 and SEQ ID NO: 79, and a second isolated polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 78, and SEQ ID NO: 80.

In one embodiment, the invention is directed to a composition that comprises a first isolated polynucleotide comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 77 and SEQ ID NO: 79, and a second isolated polynucleotide comprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 83, and SEQ ID NO: 84.

In a further aspect, the invention is directed to an isolated polynucleotide comprising a sequence that encodes a polypeptide comprising two 4-1BBL fragments comprising an amino acid sequence that is at least about 90%, 95%, 98% or 100% identical to an amino acid sequence shown in SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175 or SEQ ID NO: 183, and to a polynucleotide comprising a sequence that encodes a polypeptide comprising one 4-1BBL fragment comprising an amino acid sequence that is at least about 90%, 95%, 98% or 100% identical to an amino acid sequence shown in SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175 or SEQ ID NO: 183.

Furthermore, provided is an isolated polynucleotide encoding a OX40 ligand trimer-containing antigen binding molecule, wherein the polynucleotide comprises (a) a sequence that encodes a moiety capable of specific binding to TnC, (b) a sequence that encodes a polypeptide comprising two ectodomains of OX40L or two fragments thereof that are connected to each other by a peptide linker and (c) a sequence that encodes a polypeptide comprising one ectodomain of OX40L or a fragment thereof.

In another aspect, the invention is directed to an isolated polynucleotide comprising a sequence that encodes a polypeptide comprising two 4-1BBL fragments comprising an amino acid sequence that is at least about 90%, 95%, 98% or 100% identical to an amino acid sequence shown in SEQ ID NO: 181 or SEQ ID NO: 182, and to a polynucleotide comprising a sequence that encodes a polypeptide comprising one 4-1BBL fragment comprising an amino acid sequence that is at least about 90%, 95%, 98% or 100% identical to an amino acid sequence shown in SEQ ID NO: 181 or SEQ ID NO: 182.

In further aspects, the invention relates to the polynucleotides comprising a sequence that is at least about 90%, 95%, 98% or 100% identical to the specific cDNA sequences disclosed herein. In a particular aspect, the invention relates to a polynucleotide comprising a sequence that is identical to one of the specific cDNA sequences disclosed herein.

In other aspects, the nucleic acid molecule comprises or consists of a nucleotide sequence that encodes an amino acid sequence as set forth in any one of SEQ ID NOs: 176, 177, 184, 185, 186, 187, 188 or 189. In a further aspect, the nucleic acid molecule comprises or consists of a nucleotide sequence that encodes an amino acid sequence as set forth in any one of SEQ ID NOs: 102, 103, 104, 108, 109, 112, 113, 116, 117, 120, 121, 124, 125, 127, 130, 131, 133, 136, 137, 178 and 179.

In still other aspects, the nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, 49, 50, 51, 52, 53, 54, 61, 62, 63, 64, 65, 66, 73, 74, 75, 76, 81, 82, 88, 89, 90, 91, 96, 98, 99, 100, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126, 128, 129, 132, 134 or 135.

In certain aspects, the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). RNA of the present invention may be single stranded or double stranded.

Recombinant Methods

Antigen binding molecules of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the antigen binding molecule or polypeptide fragments thereof, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one aspect of the invention, a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the antigen binding molecule (fragment) along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the antigen binding molecule or polypeptide fragments thereof (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements. As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5′ and 3′ untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the antigen binding molecule of the invention or polypeptide fragments thereof, or variants or derivatives thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.

Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit â-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of the antigen binding molecule or polypeptide fragments thereof is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding an antigen binding molecule of the invention or polypeptide fragments thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native signal peptide, e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the fusion protein may be included within or at the ends of the polynucleotide encoding an antigen binding molecule of the invention or polypeptide fragments thereof.

In a further aspect of the invention, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one aspect, a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) an antigen binding molecule of the invention of the invention. As used herein, the term “host cell” refers to any kind of cellular system which can be engineered to generate the fusion proteins of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of antigen binding molecules are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the antigen binding molecule for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006).

Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells, e g, mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a polypeptide comprising either the heavy or the light chain of an immunoglobulin, may be engineered so as to also express the other of the immunoglobulin chains such that the expressed product is an immunoglobulin that has both a heavy and a light chain.

In one aspect, a method of producing an antigen binding molecule of the invention or polypeptide fragments thereof is provided, wherein the method comprises culturing a host cell comprising polynucleotides encoding the antigen binding molecule of the invention or polypeptide fragments thereof, as provided herein, under conditions suitable for expression of the antigen binding molecule of the invention or polypeptide fragments thereof, and recovering the antigen binding molecule of the invention or polypeptide fragments thereof from the host cell (or host cell culture medium).

In the TNF family ligand trimer-containing antigen binding molecule of the invention, the components (at least one moiety capable of specific binding to TnC, one polypeptide comprising two ectodomains of a TNF ligand family member or fragments thereof and a polypeptide comprising one ectodomain of said TNF family ligand family member or a fragment thereof) are not genetically fused to each other. The polypeptides are designed such that its components (two ectodomains of a TNF ligand family member or fragments thereof and other components such as CH or CL) are fused to each other directly or through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of the antigen binding molecules of the invention are found in the sequences provided herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion protein if desired, for example an endopeptidase recognition sequence.

In certain embodiments the moieties capable of specific binding to TnC (e.g. Fab fragments) forming part of the antigen binding molecule comprise at least an immunoglobulin variable region capable of binding to an antigen. Variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods to produce polyclonal antibodies and monoclonal antibodies are well known in the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g., U.S. Pat. No. 5,969,108 to McCafferty).

Any animal species of immunoglobulin can be used in the invention. Non-limiting immunoglobulins useful in the present invention can be of murine, primate, or human origin. If the fusion protein is intended for human use, a chimeric form of immunoglobulin may be used wherein the constant regions of the immunoglobulin are from a human. A humanized or fully human form of the immunoglobulin can also be prepared in accordance with methods well known in the art (see e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g., recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g., those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing the “guided selection” approach to FR shuffling). Particular immunoglobulins according to the invention are human immunoglobulins. Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see e.g. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions may also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variable regions may also be generated by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.

In certain aspects, the moieties capable of specific binding to a target cell antigen (e.g., Fab fragments) comprised in the antigen binding molecules of the present invention are engineered to have enhanced binding affinity according to, for example, the methods disclosed in PCT publication WO 2012/020006 or U.S. Pat. Appl. Publ. No. 2004/0132066. The ability of the antigen binding molecules of the invention to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., surface plasmon resonance technique (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays may be used to identify an antigen binding molecule that competes with a reference antibody for binding to a particular antigen. In certain embodiments, such a competing antigen binding molecule binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by the reference antigen binding molecule. Detailed exemplary methods for mapping an epitope to which an antigen binding molecule binds are provided in Morris (1996) “Epitope Mapping Protocols”, in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). In an exemplary competition assay, immobilized antigen is incubated in a solution comprising a first labeled antigen binding molecule that binds to the antigen and a second unlabeled antigen binding molecule that is being tested for its ability to compete with the first antigen binding molecule for binding to the antigen. The second antigen binding molecule may be present in a hybridoma supernatant. As a control, immobilized antigen is incubated in a solution comprising the first labeled antigen binding molecule but not the second unlabeled antigen binding molecule. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with immobilized antigen is measured. If the amount of label associated with immobilized antigen is substantially reduced in the test sample relative to the control sample, then that indicates that the second antigen binding molecule is competing with the first antigen binding molecule for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Antigen binding molecules of the invention prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the antigen binding molecule binds. For example, for affinity chromatography purification of fusion proteins of the invention, a matrix with protein A or protein G may be used. Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an antigen binding molecule essentially as described in the Examples. The purity of the antigen binding molecule or fragments thereof can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the antigen binding molecules expressed as described in the Examples were shown to be intact and properly assembled as demonstrated by reducing and non-reducing SDS-PAGE.

Assays

The antigen binding molecules provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Affinity Assays

The affinity of the antigen binding molecule provided herein for TnC and/or for the corresponding TNF receptor can be determined in accordance with the methods set forth in the Examples by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare), and receptors or target proteins such as may be obtained by recombinant expression. The affinity of the TNF family ligand trimer-containing antigen binding molecule for the target cell antigen can also be determined by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare), and receptors or target proteins such as may be obtained by recombinant expression. A specific illustrative and exemplary embodiment for measuring binding affinity is described in Example 4. According to one aspect, K_(D) is measured by surface plasmon resonance using a BIACORE® T100 machine (GE Healthcare) at 25° C.

2. Binding Assays and Other Assays

Binding of the TNF family ligand trimer-containing antigen binding molecule provided herein to the corresponding receptor expressing cells may be evaluated using cell lines expressing the particular receptor or target cell antigen, for example by flow cytometry (FACS). In one aspect, fresh peripheral blood mononuclear cells (PBMCs) expressing the TNF receptor are used in the binding assay. These cells are used directly after isolation (naïve PMBCs) or after stimulation (activated PMBCs). In another aspect, activated mouse splenocytes (expressing the TNF receptor molecule) were used to demonstrate the binding of the TNF family ligand trimer-containing antigen binding molecule of the invention to the corresponding TNF receptor expressing cells.

In a further aspect, cancer cell lines expressing TnC, were used to demonstrate the binding of the antigen binding molecules to the target cell antigen.

In another aspect, competition assays may be used to identify an antigen binding molecule that competes with a specific antibody or antigen binding molecule for binding to the target or TNF receptor, respectively. In certain embodiments, such a competing antigen binding molecule binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by a specific anti-target antibody or a specific anti-TNF receptor antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

3. Activity Assays

In one aspect, assays are provided for identifying TNF family ligand trimer-containing antigen binding molecules that bind to a specific target cell antigen and to a specific TNF receptor having biological activity. Biological activity may include, e.g., agonistic signalling through the TNF receptor on cells expressing the target cell antigen. TNF family ligand trimer-containing antigen binding molecules identified by the assays as having such biological activity in vitro are also provided.

In certain aspects, a TNF family ligand trimer-containing antigen binding molecule of the invention is tested for such biological activity. Assays for detecting the biological activity of the molecules of the invention are those described in Example 12. Furthermore, assays for detecting cell lysis (e.g. by measurement of LDH release), induced apoptosis kinetics (e.g. by measurement of Caspase 3/7 activity) or apoptosis (e.g. using the TUNEL assay) are well known in the art. In addition the biological activity of such complexes can be assessed by evaluating their effects on survival, proliferation and lymphokine secretion of various lymphocyte subsets such as NK cells, NKT-cells or 145 T-cells or assessing their capacity to modulate phenotype and function of antigen presenting cells such as dendritic cells, monocytes/macrophages or B-cells.

Pharmaceutical Compositions, Formulations and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositions comprising any of the antigen binding molecules provided herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the antigen binding molecules provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, a pharmaceutical composition comprises any of the antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.

Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more antigen binding molecules dissolved or dispersed in a pharmaceutically acceptable excipient. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one antigen binding molecule and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. In particular, the compositions are lyophilized formulations or aqueous solutions. As used herein, “pharmaceutically acceptable excipient” includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers and combinations thereof, as would be known to one of ordinary skill in the art.

Parenteral compositions include those designed for administration by injection, e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection. For injection, the antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the fusion proteins may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the fusion proteins of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein. Suitable pharmaceutically acceptable excipients include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutically acceptable excipients herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.

In addition to the compositions described previously, the fusion proteins may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the fusion proteins may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the fusion proteins of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

The antigen binding molecules may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g. those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.

The composition herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

Therapeutic Methods and Compositions

Any of the antigen binding molecules provided herein may be used in therapeutic methods. For use in therapeutic methods, antigen binding molecules of the invention can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

In one aspect, antigen binding molecules of the invention for use as a medicament are provided. In further aspects, antigen binding molecules of the invention for use in treating a disease, in particular for use in the treatment of cancer, are provided. In certain aspects, antigen binding molecules of the invention for use in a method of treatment are provided. In one aspect, the invention provides an antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof. In certain aspects, the invention provides an antigen binding molecule for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the fusion protein. In certain aspects, the disease to be treated is cancer. Examples of cancers include solid tumors, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma and acute lymphoblastic leukemia. Thus, an antigen binding molecule as described herein for use in the treatment of cancer is provided. The subject, patient, or “individual” in need of treatment is typically a mammal, more specifically a human.

In another aspect, provided is an antigen binding molecule as described herein for use in the treatment of infectious diseases, in particular for the treatment of viral infections. In a further aspect, provided is an antigen binding molecule as described herein for use in the treatment of autoimmune diseases such as for example Lupus disease.

In one aspect, provided is an antigen binding molecule according to the invention for use in treating head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC) and Mesothelioma, wherein the target cell antigen is TnC.

In a further aspect, the invention relates to the use of an antigen binding molecule in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof. In one aspect, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments the disease to be treated is a proliferative disorder, particularly cancer. Thus, in one aspect, the invention relates to the use of an antigen binding molecule of the invention in the manufacture or preparation of a medicament for the treatment of cancer. Examples of cancers include solid tumors, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma and acute lymphoblastic leukemia. Other cell proliferation disorders that can be treated using an antigen binding molecule of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. A skilled artisan may recognize that in some cases the antigen binding molecule may not provide a cure but may only provide partial benefit. In some aspects, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some aspects, an amount of antigen binding molecule that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount”.

In a further aspect, the invention relates to the use of an antigen binding molecule as described herein in the manufacture or preparation of a medicament for the treatment of infectious diseases, in particular for the treatment of viral infections or for the treatment of autoimmune diseases, for example Lupus disease.

In a further aspect, the invention provides a method for treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of an antigen binding molecule of the invention. In one aspect a composition is administered to said individual, comprising a fusion protein of the invention in a pharmaceutically acceptable form. In certain aspects, the disease to be treated is a proliferative disorder. In a particular aspect, the disease is cancer. In another aspect, the disease is an infectious disease or an autoimmune disease. In certain aspects, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g. an anti-cancer agent if the disease to be treated is cancer. An “individual” according to any of the above embodiments may be a mammal, preferably a human.

For the prevention or treatment of disease, the appropriate dosage of an antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of fusion protein, the severity and course of the disease, whether the fusion protein is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the fusion protein, and the discretion of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

The antigen binding molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of antigen binding molecule can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antigen binding molecule would be in the range from about 0.005 mg/kg to about 10 mg/kg. In other examples, a dose may also comprise from about 1 μg/kg body weight, about 5 μg/kg body weight, about 10 μg/kg body weight, about 50 μg/kg body weight, about 100 μg/kg body weight, about 200 μg/kg body weight, about 350 μg/kg body weight, about 500 μg/kg body weight, about 1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg body weight, to about 1000 mg/kg body weight or more per administration, and any range derivable therein. In examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 μg/kg body weight to about 500 mg/kg body weight etc., can be administered, based on the numbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or e.g., about six doses of the fusion protein). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

The antigen binding molecules of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the antigen binding molecules of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC₅₀ as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

Dosage amount and interval may be adjusted individually to provide plasma levels of the antigen binding molecules which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effective local concentration of the antigen binding molecule may not be related to plasma concentration. One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the antigen binding molecules described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of a fusion protein can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD₅₀ (the dose lethal to 50% of a population) and the ED₅₀ (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. Antigen binding molecules that exhibit large therapeutic indices are preferred. In one embodiment, the antigen binding molecule according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with fusion proteins of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.

Other Agents and Treatments

The antigen binding molecules of the invention may be administered in combination with one or more other agents in therapy. For instance, a fusion protein of the invention may be co-administered with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent that can be administered for treating a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an additional therapeutic agent is another anti-cancer agent.

Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of fusion protein used, the type of disorder or treatment, and other factors discussed above. The antigen binding molecules of the invention are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the antigen binding molecule of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle). At least one active agent in the composition is an antigen binding molecule of the invention.

The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antigen binding molecule of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.

Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

TABLE C (Sequences): SEQ ID NO: Name Sequence 1 Nucleotide sequence huTNC Table 3 2 Nucleotide sequence Table 3 muTNC 3 Nucleotide sequence Table 3 cynoTNC 4 huTNC Table 3 5 muTNC Table 3 6 cynoTNC Table 3 7 GST huTNC fn5 A1234 BC Table 4 fn6 B 8 GST huTNCfn5 mu A124 Table 4 BC hu fn6 B 9 GST TNC hu fn5 B-C fn6 B Table 4 10 GST huTNC fn5 A1234 fn6 B Table 4 11 huTNC A4 B Table 4 12 huTNC A1 B Table 4 13 Nucleotide sequence Table 6 pRJH33 library template DP88-4 library 14 Nucleotide sequence Fab Table 7 light chain V1_5 15 Nucleotide sequence Fab Table 7 heavy chain VH1_69 16 Fab light chain Vk1_5 Table 8 17 Fab heavy chain VH1_69 Table 8 (DP88) 18 Nucleotide sequence LMB3 Table 9 19 Nucleotide sequence Table 9 Vk1_5_L3r_S 20 Nucleotide sequence Table 9 Vk1_5_L3r_SY 21 Nucleotide sequence Table 9 Vk1_5_L3r_SPY 22 Nucleotide sequence RJH31 Table 9 23 Nucleotide sequence RJH32 Table 9 24 Nucleotide sequence DP88- Table 9 v4-4 25 Nucleotide sequence DP88- Table 9 v4-6 26 Nucleotide sequence DP88- Table 9 v4-8 27 Nucleotide sequence Table 9 fdseqlong 28 Nucleotide sequence Table 10 pRJH53 library template of lambda-DP47 library Vl3_19/VH3_23 29 Nucleotide sequence Fab Table 11 light chain Vl3_19 30 Nucleotide sequence Fab Table 11 heavy chain VH3_23 31 Fab light chain Vl3_19 Table 12 32 Fab heavy chain VH3_23 Table 12 (DP47) 33 Nucleotide sequence Table 13 Vl_3_19_L3r_V 34 Nucleotide sequence Table 13 Vl_3_19_L3r_HV 35 Nucleotide sequence Table 13 Vl_3_19_L3r_HLV 36 Nucleotide sequence RJH80 Table 13 37 Nucleotide sequence Table 13 DP47CDR3_ba (mod.) 38 Nucleotide sequence DP47- Table 13 v4-4 39 Nucleotide sequence DP47- Table 13 v4-6 40 Nucleotide sequence DP47- Table 13 v4-8 41 Nucleotide sequence Table 14 18D4 VL 42 Nucleotide sequence Table 14 18D4 VH 43 Nucleotide sequence Table 14 11C7 VL 44 Nucleotide sequence Table 14 11C7 VH 45 18D4 VL Table 15 46 18D4 VH Table 15 47 11C7 VL Table 15 48 11C7 VH Table 15 49 Nucleotide sequence Table 16 18D4 LCDR1 50 Nucleotide sequence Table 16 18D4 LCDR2 51 Nucleotide sequence Table 16 18D4 LCDR3 52 Nucleotide sequence Table 16 11C7 LCDR1 53 Nucleotide sequence Table 16 11C7 LCDR2 54 Nucleotide sequence Table 16 11C7 LCDR3 55 18D4 LCDR1 Table 17 56 18D4 LCDR2 Table 17 57 18D4 LCDR3 Table 17 58 11C7 LCDR1 Table 17 59 11C7 LCDR2 Table 17 60 11C7 LCDR3 Table 17 61 Nucleotide sequence Table 18 18D4 HCDR1 62 Nucleotide sequence Table 18 18D4 HCDR2 63 Nucleotide sequence Table 18 18D4 HCDR3 64 Nucleotide sequence Table 18 11C7 HCDR1 65 Nucleotide sequence Table 18 11C7 HCDR2 66 Nucleotide sequence Table 18 11C7 HCDR3 67 18D4 HCDR1 Table 19 68 18D4 HCDR2 Table 19 69 18D4 HCDR3 Table 19 70 11C7 HCDR1 Table 19 71 11C7 HCDR2 Table 19 72 11C7 HCDR3 Table 19 73 Nucleotide sequence Table 20 18D4 Light chain 74 Nucleotide sequence Table 20 18D4 Heavy chain 75 Nucleotide sequence Table 20 11C7 Light chain 76 Nucleotide sequence Table 20 11C7 Heavy chain 77 18D4 Light chain Table 21 78 18D4 Heavy chain Table 21 79 11C7 Light chain Table 21 80 11C7 Heavy chain Table 21 81 Nucleotide sequence Table 22 18D4 Heavy chain PGLALA 82 Nucleotide sequence 11C7 Table 22 Heavy chain PGLALA 83 18D4 Heavy chain Table 23 PGLALA 84 11C7 Heavy chain PGLALA Table 23 85 Human 4-1BB Fc(kih) Table 27 86 Cynomolgus 4-1BB Fc(kih) Table 27 87 Murine 4-1BB Fc(kih) Table 27 88 nucleotide sequence Table 28 Fc hole chain 89 nucleotide sequence Table 28 Human 4-1BB Fc(kih) 90 nucleotide sequence Table 28 Cynomolgus 4-1BB Fc(kih) 91 nucleotide sequence Table 28 Murine 4-1BB Fc(kih) 92 Fc hole chain Table 28 93 human 4-1BB antigen Fc Table 28 knob chain 94 Cynomolgus 4-1BB Fc(kih) Table 28 95 Murine 4-1BB Fc(kih) Table 28 96 nucleotide sequence Table 29 Human 4-1BB His 97 Human 4-1BB His Table 29 98 Nucleotide sequence dimeric Table 30 ligand (71-254)-CL* Fc knob chain 99 Nucleotide sequence Table 30 monomeric ligand (71-254)- CH1* 100 Nucleotide sequence anti- Table 30 TnC(18D4) Fc hole chain 101 Randomized sequence 1 NNNNNNNNNN 102 Dimeric ligand (71-254)- Table 31 CL* Fc knob chain 103 Monomeric ligand (71-254)- Table 31 CH1* 104 anti-TnC(18D4) Fc hole Table 31 chain 105 Randomized sequence 2 NNNNNNNNNNN 106 Nucleotide sequence dimeric Table 32 ligand (71-254)-CL Fc knob chain 107 Nucleotide sequence Table 32 monomeric ligand (71-254)- CH1 108 Dimeric ligand (71-254)- Table 33 CL Fc knob chain 109 Monomeric ligand (71-254)- Table 33 CH1 110 Nucleotide sequence anti- Table 34 TnC(18D4) Fc hole dimeric ligand (71-254) chain 111 Nucleotide sequence anti- Table 34 TnC(18D4) Fc knob monomeric ligand (71-254) chain 112 anti-TnC(18D4) Fc hole Table 35 dimeric ligand (71-254) chain 113 anti-TnC(18D4) Fc knob Table 35 monomeric ligand (71-254) chain 114 Nucleotide sequence dimeric Table 36 ligand (71-248)-CL* Fc knob chain 115 Nuceotide sequence Table 36 monomeric ligand (71-248)- CH1* 116 Dimeric ligand (71-248)- Table 37 CL* Fc knob chain 117 Monomeric ligand (71-248)- Table 37 CH1* 118 Nucleotide sequence dimeric Table 38 ligand (71-248)-CL Fc knob chain 119 Nucleotide sequence Table 38 monomeric ligand (71-248)- CH1 120 Dimeric ligand (71-248)- Table 39 CL Fc knob chain 121 Monomeric ligand (71-248)- Table 39 CH1 122 Nucleotide sequence anti- Table 40 TnC(18D4) Fc hole dimeric ligand (71-248) chain 123 Nucleotide sequence anti- Table 40 TnC(18D4) Fc knob monomeric (71-248) ligand 124 anti-TnC(18D4) Fc hole Table 41 dimeric ligand (71-248) chain 125 anti-TnC(18D4) Fc knob Table 41 monomeric (71-248) ligand 126 Nucleotide sequence Fc hole Table 42 dimeric ligand (71-248) chain 127 Fc hole dimeric ligand (71- Table 43 248) chain 128 Nucleotide sequence Fc hole Table 44 monomeric ligand (71-248) chain 129 Nucleotide sequence anti- Table 44 TnC(18D4) Fc knob dimeric ligand (71-248) chain 130 Fc hole monomeric ligand Table 45 (71-248) chain 131 anti-TnC(18D4) Fc knob Table 45 dimeric ligand (71-248) chain 132 Nucleotide sequence Fc Table 46 knob monomeric ligand (71- 248) chain 133 Fc knob monomeric ligand Table 47 (71-248) chain 134 Nucleotide sequence anti- Table 48 TnC(18D4) Fc hole monomeric ligand (71-248) chain 135 Nucleotide sequence Fc Table 48 knob dimeric ligand (71- 248) chain 136 anti-TnC(18D4) Fc hole Table 49 monomeric ligand (71-248) chain 137 Fc knob dimeric ligand (71- Table 49 248) chain 138 nucleotide sequence DP47 Table 50 Fc-hole chain 139 nucleotide sequence DP47 Table 50 light chain 140 DP47 Fc-hole chain Table 50 141 DP47 light chain Table 50 142 nucleotide sequence DP47 Table 51 Fc hole chain fused to dimeric hu 4-1BBL (71-254) 143 nucleotide sequence DP47 Table 51 Fc knob chain fused to monomeric hu 4-1BBL (71- 254) 144 DP47 Fc hole chain fused to Table 51 dimeric hu 4-1BBL (71-254) 145 DP47 Fc knob chain fused Table 51 to monomeric hu 4-1BBL (71-254) 146 nucleotide sequence DP47 Table 54 heavy chain (hu IgG1 PGLALA) 147 DP47 heavy chain (hu IgG1 Table 54 PGLALA) 148 nucleotide sequence anti- Table 55 TnC(18D4) heavy chain (huIgG1 PGLALA) 149 anti-TnC(18D4) heavy chain Table 55 (huIgG1 PGLALA) 150 Peptide linker (G4S)₂ GGGGSGGGGS 151 Peptide linker (SG4)₂ SGGGGSGGGG 152 Peptide linker G4(SG4)₂ GGGGSGGGGSGGGG 153 Peptide linker 1 GSPGSSSSGS 154 Peptide linker (G4S)₄ GGGGSGGGGSGGGGSGGGGS 155 Peptide linker 2 GSGSGNGS 156 Peptide linker 3 GGSGSGSG 157 Peptide linker 4 GGSGSG 158 Peptide linker 5 GGSG 159 Peptide linker 6 GGSGNGSG 160 Peptide linker 7 GGNGSGSG 161 Peptide linker 8 GGNGSG 162 Peptide linker 9 GGGGS 163 Leader Sequence 1 MDWTWRILFLVAAATGAHS 164 Leader Sequence 1 (DNA1) ATGGACTGGACCTGGAGAATCCTCTTCTTGGT GGCAGCAGCCACAGGAGCCCACTCC 165 Leader Sequence 1 (DNA2) ATGGACTGGACCTGGAGGATCCTCTTCTTGGT GGCAGCAGCCACAGGAGCCCACTCC 166 Leader Sequence 2 MDMRVPAQLLGLLLLWFPGA 167 Leader Sequence 2 (DNA) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGG CCTCCTGCTGCTCTGGTTCCCAGGTGCCAGGT GT 168 Leader Sequence 3 MGWSCIILFLVATATGVHS 169 Leader Sequence 3 (DNA 1) ATGGGATGGAGCTGTATCATCCTCTTCTTGGT AGCAACAGCTACCGGTGTGCATTCG 170 Leader Sequence 3 (DNA 2) ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTG GCTACCGCCACTGGAGTGCATTCC 171 Leader Sequence 3 (DNA 3) ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTC GCCACAGCCACCGGCGTGCACTCT 172 Human (hu) 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254) DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSE 173 hu 4-1BBL (85-254) LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA GVSLTGGLSYKEDTKELVVAKAGVYYVFFQLEL RRVVAGEGSGSVSLALHLQPLRSAAGAAALALT VDLPPASSEARNSAFGFQGRLLHLSAGQRLGVH LHTEARARHAWQLTQGATVLGLFRVTPEIPAGL PSPRSE 174 hu 4-1BBL (80-254) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYS DPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHLSAG QRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSE 175 hu 4-1BBL (52-254) PWAVSGARASPGSAASPRLREGPELSPDDPAGLL DLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAG VSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR RVVAGEGSGSVSLALHLQPLRSAAGAAALALTV DLPPASSEARNSAFGFQGRLLHLSAGQRLGVHL HTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSE 176 dimeric hu 4-1BBL (71-254) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI connected by (G4S)₂ (SEQ DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA ID NO: 150) linker KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGP ELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL SWYSDPGLAGVSLTGGLSYKEDTKELVVAKAG VYYVFFQLELRRVVAGEGSGSVSLALHLQPLRS AAGAAALALTVDLPPASSEARNSAFGFQGRLLH LSAGQRLGVHLHTEARARHAWQLTQGATVLGL FRVTPEIPAGLPSPRSE 177 monomeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254) plus (G4S)₂ (SEQ ID DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA NO: 150) linker KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGSGGGGS 178 dimeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254)-CH1 Fc knob chain DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGP ELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL SWYSDPGLAGVSLTGGLSYKEDTKELVVAKAG VYYVFFQLELRRVVAGEGSGSVSLALHLQPLRS AAGAAALALTVDLPPASSEARNSAFGFQGRLLH LSAGQRLGVHLHTEARARHAWQLTQGATVLGL FRVTPEIPAGLPSPRSEGGGGSGGGGSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALGAPIEKTISKAKGQPREPQVYTLPPCRDEL TKNQVSLWCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 179 monomeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254)-CL DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 180 hu 4-1BBL (50-254) ACPWAVSGARASPGSAASPRLREGPELSPDDPA GLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGL AGVSLTGGLSYKEDTKELVVAKAGVYYVFFQL ELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG VHLHTEARARHAWQLTQGATVLGLFRVTPEIPA GLPSPRSE 181 hu OX40L (51-183) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEI MKVQNNSVIINCDGFYLISLKGYFSQEVNISLHY QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYL NVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 182 hu OX40L (52-183) VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIM KVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQ KDEEPLFQLKKVRSVNSLMVASLTYKDKVYLN VTTDNTSLDDFHVNGGELILIHQNPGEFCVL 183 Human (hu) 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 248) DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGL 184 dimeric hu 4-1BBL (71-248) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI connected by (G4S)₂ (SEQ DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA ID NO: 150) linker KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDD PAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDP GLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAA LALTVDLPPASSEARNSAFGFQGRLLHLSAGQRL GVHLHTEARARHAWQLTQGATVLGLFRVTPEIP AGL 185 dimeric hu 4-1BBL (80-254) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYS connected by (G4S)₂ (SEQ DPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV ID NO: 150) linker FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHLSAG QRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSEGGGGSGGGGSDPAGLLDLRQG MFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGG LSYKEDTKELVVAKAGVYYVFFQLELRRVVAG EGSGSVSLALHLQPLRSAAGAAALALTVDLPPA SSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 186 dimeric hu 4-1BBL (52-254) PWAVSGARASPGSAASPRLREGPELSPDDPAGLL connected by (G4S)₂ (SEQ DLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAG ID NO: 150) linker VSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR RVVAGEGSGSVSLALHLQPLRSAAGAAALALTV DLPPASSEARNSAFGFQGRLLHLSAGQRLGVHL HTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSEGGGGSGGGGSPWAVSGARASPGSAASPR LREGPELSPDDPAGLLDLRQGMFAQLVAQNVLL IDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVV AKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQG RLLHLSAGQRLGVHLHTEARARHAWQLTQGAT VLGLFRVTPEIPAGLPSPRSE 187 monomeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254) plus (G4S)₁ (SEQ ID DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA NO: 162) linker KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGS 188 monomeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 248) plus (G4S)₂ (SEQ ID DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA NO: 150) linker KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLGGGGSGGGGS 189 monomeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 248) plus (G4S)₁ (SEQ ID DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA NO: 162) linker KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLGGGGS 190 dimeric huOX40L (51-183) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEI connected by (G4S)₂ (SEQ MKVQNNSVIINCDGFYLISLKGYFSQEVNISLHY ID NO: 150) linker QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYL NVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGG GGSGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFI LTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQN PGEFCVL 191 dimeric huOX40L (52-183) VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIM connected by (G4S)2 (SEQ KVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQ ID NO: 150) linker KDEEPLFQLKKVRSVNSLMVASLTYKDKVYLN VTTDNTSLDDFHVNGGELILIHQNPGEFCVLGGG GSGGGGSVSHRYPRIQSIKVQFTEYKKEKGFILTS QKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEV NISLHYQKDEEPLFQLKKVRSVNSLMVASLTYK DKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEF CVL 192 hu 4-1BBL (85-248) LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA GVSLTGGLSYKEDTKELVVAKAGVYYVFFQLEL RRVVAGEGSGSVSLALHLQPLRSAAGAAALALT VDLPPASSEARNSAFGFQGRLLHLSAGQRLGVH LHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 193 hu 4-1BBL (80-248) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYS DPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHLSAG QRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGL 194 hu 4-1BBL (52-248) PWAVSGARASPGSAASPRLREGPELSPDDPAGLL DLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAG VSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR RVVAGEGSGSVSLALHLQPLRSAAGAAALALTV DLPPASSEARNSAFGFQGRLLHLSAGQRLGVHL HTEARARHAWQLTQGATVLGLFRVTPEIPAGL 195 Dimeric hu OX40L (51-183)- QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEI CL* Fc knob chain MKVQDNSVIINCDGFYLISLKGYFSQEVDISLHY QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYL NVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGG GGSGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFI LTSQKEDEIMKVQDNSVIINCDGFYLISLKGYFS QEVDISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQN PGEFCVLGGGGSGGGGSRTVAAPSVFIFPPSDRK LKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGECDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPCRDELTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 196 Monomeric hu OX40L (51- QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEI 183)-CH1* MKVQDNSVIINCDGFYLISLKGYFSQEVDISLHY QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYL NVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGG GGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALG CLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDEKVEPKSC 197 human TnC UniProt no. P24821 198 human MCSP UniProt no. Q6UVK1 199 human EGFR UniProt no. P00533 200 human CD19 UniProt no. P15391 201 human CD20 Uniprot no. P11836 202 human CD33 UniProt no. P20138 203 human Lymphotoxin α UniProt no. P01374 204 human TNF UniProt no. P01375 205 human Lymphotoxin β UniProt no. Q06643 206 human OX40L UniProt no. P23510 207 human CD40L UniProt no. P29965 208 human FasL UniProt no. P48023 209 human CD27L UniProt no. P32970 210 human CD30L UniProt no. P32971 211 human 4-1BBL UniProt no. P41273 212 human TRAIL UniProt no. P50591 213 human RANKL UniProt no. O14788 214 human TWEAK UniProt no. O43508 215 human APRIL UniProt no. O75888 216 human BAFF UniProt no. Q9Y275 217 human LIGHT UniProt no. O43557 218 human TL1A UniProt no. O95150 219 human GITRL UniProt no. Q9UNG2 220 human ectodysplasin A UniProt no. Q92838

General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Amino acids of antibody chains are numbered and referred to according to the EU numbering systems according to Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) as defined above.

Particular Embodiments

-   1. A TNF family ligand trimer-containing antigen binding molecule     comprising     -   (a) at least one moiety capable of specific binding to         Tenascin-C (TnC) and     -   (b) a first and a second polypeptide that are linked to each         other by a disulfide bond,     -   wherein the antigen binding molecule is characterized in that         the first polypeptide comprises two ectodomains of a TNF ligand         family member or a fragment thereof that are connected to each         other by a peptide linker and in that the second polypeptide         comprises only one ectodomain of said TNF ligand family member         or a fragment thereof. -   2. A TNF family ligand trimer-containing antigen binding molecule     comprising     -   (a) at least one antigen binding moiety capable of specific         binding to Tenascin-C (TnC) and     -   (b) a first and a second polypeptide that are linked to each         other by a disulfide bond,     -   wherein the antigen binding molecule is characterized in that         the first polypeptide comprises two ectodomains of a TNF ligand         family member or a fragment thereof that are connected to each         other by a peptide linker and in that the second polypeptide         comprises only one ectodomain of said TNF ligand family member         or a fragment thereof. -   3. The TNF family ligand trimer-containing antigen binding molecule     of embodiment 1 or 2, further comprising     -   (c) an Fc domain composed of a first and a second subunit         capable of stable association. -   4. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 3, comprising     -   (a) at least one moiety capable of specific binding to TnC and     -   (b) a first and a second polypeptide that are linked to each         other by a disulfide bond,     -   wherein the antigen binding molecule is characterized in that         -   (i) the first polypeptide contains a CH1 or CL domain and             the second polypeptide contains a CL or CH1 domain,             respectively, wherein the second polypeptide is linked to             the first polypeptide by a disulfide bond between the CH1             and CL domain, and wherein the first polypeptide comprises             two ectodomains of a TNF ligand family member or fragments             thereof that are connected to each other and to the CH1 or             CL domain by a peptide linker and wherein the second             polypeptide comprises one ectodomain of said TNF ligand             family member or a fragment thereof connected via a peptide             linker to the CL or CH1 domain of said polypeptide, or         -   (ii) the first polypeptide contains a CH3 domain and the             second polypeptide contains a CH3 domain, respectively, and             wherein the first polypeptide comprises two ectodomains of a             TNF ligand family member or fragments thereof that are             connected to each other and to the C-terminus of the CH3             domain by a peptide linker and wherein the second             polypeptide comprises only one ectodomain of said TNF ligand             family member or a fragment thereof connected via a peptide             linker to C-terminus of the CH3 domain of said polypeptide,             or         -   (iii) the first polypeptide contains a VH-CL or a VL-CH1             domain and the second polypeptide contains a VL-CH1 domain             or a VH-CL domain, respectively, wherein the second             polypeptide is linked to the first polypeptide by a             disulfide bond between the CH1 and CL domain, and wherein             the first polypeptide comprises two ectodomains of a TNF             ligand family member or fragments thereof that are connected             to each other and to to VH or VL by a peptide linker and             wherein the second polypeptide comprises one ectodomain of             said TNF ligand family member or a fragment thereof             connected via a peptide linker to VL or VH of said             polypeptide. -   5. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 4, wherein the TNF ligand family     member costimulates human T-cell activation. -   6. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 5, wherein the TNF ligand family     member is selected from 4-1BBL and OX40L. -   7. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 6, wherein the TNF ligand family     member is 4-1BBL. -   8. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 7, wherein the ectodomain of a TNF     ligand family member comprises the amino acid sequence selected from     the group consisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO:     174, SEQ ID NO: 175, SEQ ID NO: 183, SEQ ID NO: 192, SEQ ID NO: 193     and SEQ ID NO: 194, particularly the amino acid sequence of SEQ ID     NO: 172 or SEQ ID NO: 183. -   9. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 8, wherein the ectodomain of a TNF     ligand family member comprises the amino acid sequence of SEQ ID NO:     183. -   10. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 9, comprising     -   (a) at least one moiety capable of specific binding to TnC and     -   (b) a first and a second polypeptide that are linked to each         other by a disulfide bond,     -   wherein the antigen binding molecule is characterized in that         the first polypeptide comprises the amino acid sequence selected         from the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ         ID NO: 185 and SEQ ID NO: 186 and in that the second polypeptide         comprises the amino acid sequence selected from the group         consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and         SEQ ID NO: 183. -   11. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 10, comprising     -   (a) at least one moiety capable of specific binding to TnC, and     -   (b) a first polypeptide containing a CH1 or CL domain and a         second polypeptide containing a CL or CH1 domain, respectively,         wherein the second polypeptide is linked to the first         polypeptide by a disulfide bond between the CH1 and CL domain,     -   and wherein the antigen binding molecule is characterized in         that the first polypeptide comprises two ectodomains of a TNF         ligand family member or fragments thereof that are connected to         each other and to the CH1 or CL domain by a peptide linker and         in that the second polypeptide comprises only one ectodomain of         said TNF ligand family member or a fragment thereof connected         via a peptide linker to the CL or CH1 domain of said         polypeptide. -   12. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 11, wherein the moiety capable of     specific binding to TnC is selected from the group consisting of an     antibody fragment, a Fab molecule, a crossover Fab molecule, a     single chain Fab molecule, a Fv molecule, a scFv molecule, a single     domain antibody, an aVH and a scaffold antigen binding protein. -   13. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 12, wherein the molecule comprises     one or two moieties capable of specific binding to TnC. -   14. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 13, wherein the moiety capable of     specific binding to TnC is a Fab molecule capable of specific     binding to TnC. -   15. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 14, wherein the moiety capable of     specific binding to TnC comprises a VH domain comprising (i) CDR-H1     comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO:     70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68     or SEQ ID NO: 71, and (iii) CDR-H3 comprising the amino acid     sequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VL domain     comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID     NO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising the amino acid     sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi) CDR-L3     comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO:     60. -   16. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 15, wherein the moiety capable of     specific binding to TnC comprises a variable heavy chain comprising     an amino acid sequence of SEQ ID NO: 46 and a variable light chain     comprising an amino acid sequence of SEQ ID NO: 45 or wherein the     moiety capable of specific binding to TNC comprises a variable heavy     chain comprising an amino acid sequence of SEQ ID NO: 48 and a     variable light chain comprising an amino acid sequence of SEQ ID NO:     47. -   17. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 3 to 16, wherein the Fc domain is an IgG,     particularly an IgG1 Fc domain or an IgG4 Fc domain. -   18. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 3 to 17, wherein the Fc domain is an IgG1     Fc domain comprising the amino acid substitutions at positions 234     and 235 (EU numbering) and/or 329 (EU numbering). -   19. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 18, wherein the antigen binding     molecule comprises a first heavy chain and a first light chain, both     comprising a Fab molecule capable of specific binding to TnC, a     first peptide comprising two ectodomains of a TNF ligand family     member or fragments thereof connected to each other by a first     peptide linker fused at its C-terminus by a second peptide linker to     a second heavy or light chain, and a second peptide comprising one     ectodomain of said TNF ligand family member fused at its C-terminus     by a third peptide linker to a second light or heavy chain,     respectively. -   20. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 19, wherein the first peptide     comprising two ectodomains of a TNF ligand family member or     fragments thereof connected to each other by a first peptide linker     is fused at its C-terminus by a second peptide linker to a CH1     domain that is part of a heavy chain, and the second peptide     comprising one ectodomain of said TNF ligand family member or a     fragment thereof is fused at its C-terminus by a third peptide     linker to a CL domain that is part of a light chain. -   21. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 20, wherein the first peptide     comprising two ectodomains of a TNF ligand family member or     fragments thereof connected to each other by a first peptide linker     is fused at its C-terminus by a second peptide linker to a CL domain     that is part of a heavy chain, and the second peptide comprising one     ectodomain of said TNF ligand family member or a fragment thereof is     fused at its C-terminus by a third peptide linker to a CH1 domain     that is part of a light chain. -   22. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 21, wherein the first peptide     comprising two ectodomains of a TNF ligand family member or     fragments thereof connected to each other by a first peptide linker     is fused at its C-terminus by a second peptide linker to a VH domain     that is part of a heavy chain, and the second peptide comprising one     ectodomain of said TNF ligand family member or a fragment thereof is     fused at its C-terminus by a third peptide linker to a VL domain     that is part of a light chain. -   23. The TNF family ligand trimer-containing antigen binding molecule     of embodiments 20 or 21, wherein in the CL domain adjacent to the     TNF ligand family member the amino acid at position 123 (EU     numbering) has been replaced by arginine (R) and the amino acid at     position 124 (EU numbering) has been substituted by lysine (K), and     wherein in the CH1 domain adjacent to the TNF ligand family member     the amino acids at position 147 (EU numbering) and at position 213     (EU numbering) have been substituted by glutamic acid (E). -   24. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 23, wherein the antigen binding     molecule comprises     -   (a) a first heavy chain and a first light chain, both comprising         a Fab molecule capable of specific binding to TnC,     -   (b) a second heavy chain comprising an amino acid sequence         selected from the group consisting of SEQ ID NO: 176, SEQ ID NO:         184, SEQ ID NO: 185 and SEQ ID NO: 186, and a second light chain         comprising an amino acid sequence selected from the group         consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and         SEQ ID NO: 183. -   25. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 20, wherein the antigen binding     molecule comprises     -   (i) a first heavy chain comprising the VH domain comprising the         amino acid sequence of SEQ ID NO:46 and a first light chain         comprising the VL domain comprising the amino acid sequence of         SEQ ID NO: 45 or     -   a first heavy chain comprising the VH domain comprising the         amino acid sequence of SEQ ID NO: 48 and a first light chain         comprising the VL domain comprising the amino acid sequence of         SEQ ID NO: 47,     -   (ii) a second heavy chain comprising the amino acid sequence of         SEQ ID NO: 178, and     -   (iii) a second light chain comprising the amino acid sequence of         SEQ ID NO: 179. -   26. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 19 and 21, wherein the antigen     binding molecule comprises     -   (i) a first heavy chain comprising the VH domain comprising the         amino acid sequence of SEQ ID NO: 46 and a first light chain         comprising the VL domain comprising the amino acid sequence of         SEQ ID NO: 45 or     -   a first heavy chain comprising the VH domain comprising the         amino acid sequence of SEQ ID NO: 48 and a first light chain         comprising the VL domain comprising the amino acid sequence of         SEQ ID NO: 47,     -   (ii) a second heavy chain comprising the amino acid sequence         selected from the group consisting of SEQ ID NO: 102, SEQ ID NO:         108, SEQ ID NO: 116 and SEQ ID NO: 120, and     -   (iii) a second light chain comprising the amino acid sequence         selected from the group consisting of SEQ ID NO: 103, SEQ ID NO:         109, SEQ ID NO: 117 and SEQ ID NO: 121. -   27. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 18, comprising     -   (a) at least one moiety capable of specific binding TnC, and     -   (b) a first and a second polypeptide that are linked to each         other by a disulfide bond, wherein the antigen binding molecule         is characterized in that the first polypeptide contains a CH3         domain and the second polypeptide contains a CH3 domain,         respectively, and wherein the first polypeptide comprises two         ectodomains of a TNF ligand family member or fragments thereof         that are connected to each other and to the C-terminus of the         CH3 domain by a peptide linker and wherein the second         polypeptide comprises one ectodomain of said TNF ligand family         member or a fragment thereof connected via a peptide linker to         C-terminus of the CH3 domain of said polypeptide. -   28. The TNF family ligand trimer-containing antigen binding molecule     of embodiment 27, wherein the antigen binding molecule comprises     -   (i) a first heavy chain comprising the amino acid sequence of         SEQ ID NO: 127, a second heavy chain comprising the amino acid         sequence of SEQ ID NO: 125, and one light chain comprising the         amino acid sequence of SEQ ID NO: 77, or     -   (ii) a first heavy chain comprising the amino acid sequence of         SEQ ID NO: 130, a second heavy chain comprising the amino acid         sequence of SEQ ID NO: 131, and one light chain comprising the         amino acid sequence of SEQ ID NO: 77, or     -   (ii) a first heavy chain comprising the amino acid sequence of         SEQ ID NO: 124, a second heavy chain comprising the amino acid         sequence of SEQ ID NO: 133, and one light chain comprising the         amino acid sequence of SEQ ID NO: 77, or     -   (ii) a first heavy chain comprising the amino acid sequence of         SEQ ID NO: 136, a second heavy chain comprising the amino acid         sequence of SEQ ID NO: 137, and one light chain comprising the         amino acid sequence of SEQ ID NO: 77. -   29. The TNF family ligand trimer-containing antigen binding molecule     of embodiment 27, comprising two moieties capable of specific     binding to a target cell antigen. -   30. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 27 or 29, wherein the antigen binding     molecule comprises     -   (i) a first heavy chain comprising the amino acid sequence of         SEQ ID NO: 112, a second heavy chain comprising the amino acid         sequence of SEQ ID NO: 113, and two light chains comprising the         amino acid sequence of SEQ ID NO: 77, or     -   (ii) a first heavy chain comprising the amino acid sequence of         SEQ ID NO: 124, a second heavy chain comprising the amino acid         sequence of SEQ ID NO: 125, and two light chains comprising the         amino acid sequence of SEQ ID NO: 77. -   31. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 6, 11 to 23 and 27 to 29, wherein the     TNF ligand family member is OX40L. -   32. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 6, 11 to 23 and 27 to 29, wherein the     ectodomain of a TNF ligand family member comprises the amino acid     sequence of SEQ ID NO: 181 or SEQ ID NO: 182, particularly the amino     acid sequence of SEQ ID NO: 181. -   33. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 6, 11 to 23, 27 to 29, 31 and 32,     comprising     -   (a) at least one moiety capable of specific binding to a target         cell antigen and     -   (b) a first and a second polypeptide that are linked to each         other by a disulfide bond,     -   wherein the antigen binding molecule is characterized in that         the first polypeptide comprises the amino acid sequence of SEQ         ID NO: 190 or SEQ ID: 191 and in that the second polypeptide         comprises the amino acid sequence of SEQ ID NO: 181 or SEQ ID         NO: 182. -   34. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 6, 11 to 23, 27 to 29 and 31 to 33,     wherein the target cell antigen is Tenascin-C (TnC) and the moiety     capable of specific binding to TnC comprises a VH domain     comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID     NO: 67 or SEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid     sequence of SEQ ID NO: 68 or SEQ ID NO: 71, and (iii) CDR-H3     comprising the amino acid sequence of SEQ ID NO: 69 or SEQ ID NO:     72, and a VL domain comprising (iv) CDR-L1 comprising the amino acid     sequence of SEQ ID NO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising     the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi)     CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID     NO: 60. -   35. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 6, 11 to 23, 27 to 29 and 31 to 33,     wherein the antigen binding moiety capable of specific binding to     TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino     acid sequence of SEQ ID NO: 67, (ii) CDR-H2 comprising the amino     acid sequence of SEQ ID NO: 68, and (iii) CDR-H3 comprising the     amino acid sequence of SEQ ID NO: 69, and a VL domain     comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID     NO: 55, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:     56, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:     57. -   36. TNF family ligand trimer-containing antigen binding molecule of     any one of embodiments 1 to 6, 11 to 23, 27 to 29 and 31 to 33,     wherein the antigen binding moiety capable of specific binding to     TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino     acid sequence of SEQ ID NO: 70, (ii) CDR-H2 comprising the amino     acid sequence of SEQ ID NO: 71, and (iii) CDR-H3 comprising the     amino acid sequence of SEQ ID NO: 72, and a VL domain     comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID     NO: 58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:     59, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:     60. -   37. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 6, 11 to 23, 27 to 29 and 31 to 36,     wherein the antigen binding molecule comprises     -   (i) a first heavy chain comprising the VH domain comprising the         amino acid sequence of SEQ ID NO: 46 and a first light chain         comprising the VL domain comprising the amino acid sequence of         SEQ ID NO: 45 or     -   a first heavy chain comprising the VH domain comprising the         amino acid sequence of SEQ ID NO: 48 and a first light chain         comprising the VL domain comprising the amino acid sequence of         SEQ ID NO: 47,     -   (ii) a second heavy chain comprising the amino acid sequence         selected from the group consisting of SEQ ID NO: 195, and     -   (iii) a second light chain comprising the amino acid sequence of         SEQ ID NO: 196. -   38. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 37, wherein the antigen binding     moiety has an improved affinity. -   39. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 38, wherein the antigen binding     moiety binds to human TnC with a K_(D) value lower than about 1 nM. -   40. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 39, wherein the antigen binding     moiety has cross-species reactivity. -   41. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 40, wherein the antigen binding     moiety binds to at least one of human, mouse and cynomolgus TnC with     a K_(D) value lower than about 2 nM. -   42. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 41, wherein the antigen binding     moiety binds to human, mouse and cynomolgus TnC. -   43. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 40 to 42, wherein the antigen binding     moiety binds to the target antigen from all indicated species with     similar affinity. -   44. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 40 to 43, wherein the antigen binding     moiety binds to the target antigen from all indicated species with     similar affinity, in particular within a K_(D) range of a factor of     100, within a K_(D) range of a factor of 50, within a K_(D) range of     a factor of 20, within a K_(D) range of a factor of 10, within a     K_(D) range of a factor of 5. -   45. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 40 to 44, wherein the antigen binding     moiety binds to the target antigen from all indicated species with     similar affinity within a K_(D) range of a factor of 10. -   46. An isolated polynucleotide encoding the TNF family ligand     trimer-containing antigen binding molecule of any one of embodiments     1 to 45. -   47. A vector, particularly an expression vector, comprising the     isolated polynucleotide of embodiment 46. -   48. A host cell comprising the isolated polynucleotide of embodiment     46 or the vector of embodiment 47. -   49. A method for producing the TNF family ligand trimer-containing     antigen binding molecule of any one of embodiments 1 to 45,     comprising the steps of     -   (i) culturing the host cell of embodiment 48 under conditions         suitable for expression of the antigen binding molecule, and     -   (ii) recovering the antigen binding molecule. -   50. A pharmaceutical composition comprising the TNF family ligand     trimer-containing antigen binding molecule of any one of embodiments     1 to 45 and at least one pharmaceutically acceptable excipient. -   51. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 45, or the pharmaceutical composition     of embodiment 50, for use as a medicament. -   52. The TNF family ligand trimer-containing antigen binding molecule     of any one of embodiments 1 to 45, or the pharmaceutical composition     of embodiment 50, for use in the treatment of cancer. -   53. Use of the TNF family ligand trimer-containing antigen binding     molecule of any one of embodiments 1 to 45 for the manufacture of a     medicament for the treatment of cancer. -   54. A method of treating a disease in an individual, comprising     administering to said individual a therapeutically effective amount     of a composition comprising a TNF family ligand trimer-containing     antigen binding molecule of any one of embodiments 1 to 45 in a     pharmaceutically acceptable form. -   55. The method of embodiment 54, wherein said disease is cancer.

EXAMPLES

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturer's instructions. DNA sequences were determined by double strand sequencing. In some cases desired gene segments were prepared by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. The gene segments which are flanked by singular restriction endonuclease cleavage sites were cloned into indicated plasmids. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene Segments were designed with suitable restriction sites to allow sub-cloning into the respective expression vectors.

General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242. For expression, all constructs contained a 5′-end DNA sequence coding for a leader peptide which targets proteins for secretion in eukaryotic cells. Exemplary leader peptides and polynucleotide sequences encoding them are given in SEQ ID NO 163 to SEQ ID NO 171.

Cell Culture Techniques

Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc.

Protein Purification

Proteins were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a Protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 2.8 followed by immediate neutralization of the sample. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomeric antibody fractions were pooled, concentrated (if required) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at −20° C. or −80° C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5 on an Agilent HPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2×PBS on a Dionex HPLC-System. The eluted protein was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.

Mass Spectrometry

This section describes the characterization of the multispecific antibodies with VH/VL exchange (VH/VL CrossMabs) with emphasis on their correct assembly. The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact CrossMabs and deglycosylated/plasmin digested or alternatively deglycosylated/limited LysC digested CrossMabs.

The VH/VL CrossMabs were deglycosylated with N-Glycosidase F in a phosphate or Tris buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/ml. The plasmin or limited LysC (Roche) digestions were performed with 100 μg deglycosylated VH/VL CrossMabs in a Tris buffer pH 8 at room temperature for 120 hours and at 37° C. for 40 min, respectively. Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

Determination of Binding and Binding Affinity of Multispecific Antibodies to the Respective Antigens Using Surface Plasmon Resonance (SPR) (BIACORE)

Binding of the generated antibodies to the respective antigens is investigated by surface plasmon resonance using a BIACORE instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements Goat-Anti-Human IgG, JIR 109-005-098 antibodies are immobilized on a CMS chip via amine coupling for presentation of the antibodies against the respective antigen. Binding is measured in HBS buffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25° C. (or alternatively at 37° C.). Antigen (R&D Systems or in house purified) was added in various concentrations in solution. Association was measured by an antigen injection of 80 seconds to 3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3-10 minutes and a KD value was estimated using a 1:1 Langmuir binding model. Negative control data (e.g. buffer curves) are subtracted from sample curves for correction of system intrinsic baseline drift and for noise signal reduction. The respective Biacore Evaluation Software is used for analysis of sensorgrams and for calculation of affinity data.

Example 1

TnC Antigen Sequences and Production of Antigens

All constructs of Table 3 and Table 4 are fused to the C-term of GST and are expressed in E. coli BL21(DE3). For site specific biotinylation, the Avi-tag was added to the C-term of the Tenascin sequence, and the BirA biotin ligase was coexpressed on a separate plasmid (Avidity, Colo., USA). Growth medium was 2YT with 100 μg/ml ampicillin and 20 μg/ml chloramphenicol. Biotin was added to a final concentration of 50 μM. Protein Expression was induced with 1 mM IPTG at 22° C. overnight. Cells were harvested by centrifugation, and cell-lysis was performed by sonication in the presence of B-PER reagent (pierce 78260), and 1 mg/ml lysozyme (Sigma L6876). Lysate was centrifuged and cleared lysate was loaded on Glutathione Sepharose columns (GE Healthcare; Product No 17-0756-01). After washing, the TnC molecules were cleft from the GST via Thrombin (Sigma Aldrich; Product No 10602400001) over night at 4° C. Elution was performed in 50 mM Tris buffer pH 8.0; 200 mM NaCl, 5 mM MgCl2, 1 mM DTT and 10% glycerol. The final purification step was on a gelfiltration column (Superdex 75 16/60; GE Healthcare). Samples were flash frozen in liquid nitrogen until processing.

TABLE 3 Sequences of TnC antigens used for cross-species affinity determination (reference Table 26) Antigen Sequence SEQ ID NO huTNC ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCT SEQ ID NO: 1 TGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAG AAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGG TGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTG GAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTT AAATTAACACAGTCTATGGCCATCATACGTTATATAGC TGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAG CGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGA TATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAG ACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTA CCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCA TAAAACATATTTAAATGGTGATCATGTAACCCATCCTG ACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACA TGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTT TGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGA TAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTT TGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCAT CCTCCAAAATCGGATGGTTCAACTAGTGGTTCTGGTCA TCACCATCACCATCACTCCGCGGGTCTGGTGCCACGCG GTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGCT AAATTCGAACGCCAGCACATGGACAGCCCAGATCTGG GTACCGGTGGTGGCTCCGGTATTGAGGGACGCGGGTCC ATGGGATATCGGGGATCCGAGCTGGACACCCCCAAGG ACCTGCAGGTGTCCGAGACAGCCGAGACAAGCCTGAC CCTGCTGTGGAAAACCCCCCTGGCCAAGTTCGACCGGT ACAGACTGAACTACAGCCTGCCCACTGGACAGTGGGT CGGCGTGCAGCTGCCCCGGAACACCACCTCCTACGTGC TGCGGGGCCTGGAACCCGGCCAGGAATACAACGTCCT GCTGACGGCCGAGAAGGGCCGGCACAAGAGCAAGCCC GCCAGAGTGAAGGCCAGCACCGAGCAGGCCCCCGAGC TGGAAAACCTGACCGTGACCGAAGTGGGCTGGGACGG CCTGCGGCTGAACTGGACCGCGGCTGACCAGGCCTATG AGCACTTTATCATTCAGGTGCAGGAGGCCAACAAGGT GGAGGCAGCTCGGAACCTCACCGTGCCTGGCAGCCTTC GGGCTGTGGACATACCGGGCCTCAAGGCTGCTACGCCT TATACAGTCTCCATCTATGGGGTGATCCAGGGCTATAG AACACCAGTGCTCTCTGCTGAGGCCTCCACAGGCGAAA CACCGAACCTGGGCGAAGTGGTGGTGGCGGAAGTGGG TTGGGATGCGCTGAAACTGAACTGGACCGCGCCGGAA GGCGCGTATGAATATTTTTTCATCCAGGTGCAGGAAGC GGATACCGTTGAAGCGGCGCAGAACCTGACCGTTCCG GGCGGTCTGCGTAGCACCGATCTGCCGGGCCTGAAAG CGGCGACCCATTATACCATTACCATCCGTGGGGTGACC CAGGACTTCTCTACCACCCCTCTGAGCGTGGAGGTGCT GACCGAGGAGGTACCCGACATGGGCAACCTGACCGTG ACCGAGGTGTCCTGGGACGCCCTGCGGCTGAACTGGA CCACCCCCGACGGCACCTACGACCAGTTCACAATCCAG GTGCAGGAAGCCGACCAGGTGGAAGAAGCACATAATC TGACCGTTCCGGGTAGCCTGCGTAGCATGGAAATTCCG GGTCTGCGTGCAGGCACCCCGTATACCGTTACCCTGCA TGGTGAAGTTCGTGGTCATAGCACCCGTCCGCTGGCAG TTGAAGTTGTTACCGAAGATCTGCCGCAGCTGGGTGAT CTGGCAGTTAGCGAAGTTGGTTGGGATGGTCTGCGTCT GAATTGGACCGCAGCAGATAATGCATATGAACATTTTG TGATCCAGGTGCAAGAGGTGAATAAAGTTGAAGCAGC CCAGAATCTGACCCTGCCTGGTTCACTGCGTGCAGTTG ATATTCCGGGACTCGAGGCAGCAACCCCGTATCGTGTT AGCATTTATGGTGTTATTCGCGGTTATCGTACACCGGT TCTGAGCGCAGAAGCAAGCACCGCAAAAGAACCGGAA ATTGGTAATCTGAACGTGAGCGATATTACACCGGAATC ATTTAATCTGAGCTGGATGGCAACCGATGGTATTTTTG AAACCTTTACCATCGAGATCATCGATAGCAATCGTCTG CTGGAAACCGTGGAATATAATATTAGCGGTGCAGAAC GTACCGCACATATTAGCGGTCTGCCTCCGAGCACCGAT TTTATTGTTTATCTGAGCGGTCTGGCACCGAGCATTCGT ACCAAAACCATTAGCGCAACCGCAACCACCGAAGCAC TGCCGCTGCTGGAAAATCTGACCATTAGCGATATTAAC CCGTATGGTTTTACCGTTTCATGGATGGCAAGCGAAAA TGCATTTGATAGCTTTCTGGTTACAGTTGTGGATAGCG GTAAACTGCTGGACCCGCAAGAATTTACCCTGAGCGGC ACCCAGCGCAAACTGGAACTGCGTGGTCTGATTACCGG TATTGGTTATGAAGTTATGGTGAGCGGTTTTACCCAGG GTCATCAGACCAAACCGCTGCGTGCAGAAATTGTTACC GAAGCAATGGGTAGCCCGAAAGAAGTTATTTTTTCCGA TATCACCGAGAATTCGGCAACCGTTAGCTGGCGTGCAC CGACCGCACAGGTTGAAAGCTTTCGTATTACCTATGTT CCGATTACCGGTGGCACCCCGAGCATGGTTACAGTTGA TGGCACCAAAACCCAGACCCGTCTGGTTAAACTGATTC CGGGTGTTGAATATCTGGTTAGCATTATTGCCATGAAA GGCTTTGAAGAAAGCGAACCGGTTAGCGGTAGCTTTAC CACAGCTAGCGGCCTGAACGACATCTTCGAGGCTCAG AAAATCGAATGGCACGAAGGTACCCATCACCATCACC ACCACTAA muTNC TATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCC SEQ ID NO: 2 TTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAA GAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAG GTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTT GGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGT TAAATTAACACAGTCTATGGCCATCATACGTTATATAG CTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGA GCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGG ATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAA GACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCT ACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTC ATAAAACATATTTAAATGGTGATCATGTAACCCATCCT GACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATAC ATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGT TTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTG ATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCT TTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCA TCCTCCAAAATCGGATGGTTCAACTAGTGGTTCTGGTC ATCACCATCACCATCACTCCGCGGGTCTGGTGCCACGC GGTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGC TAAATTCGAACGCCAGCACATGGACAGCCCAGATCTG GGTACCGGTGGTGGCTCCGGTATTGAGGGACGCGGGT CCATGGGATATCGGGGATCCGAGCTGGACACCCCCAA GGACCTGCAGGTGTCCGAGACAGCCGAGACAAGCCTG ACCCTGCTGTGGAAAACCCCCCTGGCCAAGTTCGACCG GTACAGACTGAACTACAGCCTGCCCACTGGACAGTGG GTCGGCGTGCAGCTGCCCCGGAACACCACCTCCTACGT GCTGCGGGGCCTGGAACCCGGCCAGGAATACAACGTC CTGCTGACGGCCGAGAAGGGCCGGCACAAGAGCAAGC CCGCCAGAGTGAAGGCCAGCACCGAGGAAGTGCCCAG CCTGGAAAACCTGACCGTGACCGAGGCCGGCTGGGAC GGCCTGCGGCTGAACTGGACCGCCGACGACCTGGCCT ACGAGTACTTCGTGATCCAGGTGCAGGAAGCCAACAA CGTCGAGACAGCCCACAACTTCACCGTGCCCGGCAACC TGAGAGCCGCCGACATCCCCGGCCTGAAGGTGGCCAC ATCCTACCGGGTGTCCATCTACGGCGTGGCCAGGGGCT ACCGGACCCCCGTGCTGTCCGCCGAGACAAGCACCGG CACCACGCCGAACCTGGGCGAAGTGACCGTGGCGGAA GTGGGTTGGGATGCGCTGACCCTGAATTGGACCGCACC GGAAGGCGCGTATAAAAACTTTTTCATCCAGGTGCTGG AAGCGGATACCACCCAGACCGTGCAGAACCTGACCGT GCCGGGTGGTCTGCGTAGCGTAGATCTGCCTGGTCTGA AAGCAGCAACCCGCTATTACATTACCCTGCGTGGTGTT ACCCAGGATTTTGGCACCGCACCGCTGAGCGTTGAAGT TCTGACCGAGGATCTGCCGCAGCTGGGTGGTCTGAGCG TTACCGAAGTTAGTTGGGATGGTCTGACCCTGAATTGG ACCACCGATGATCTGGCATATAAACATTTTGTGGTGCA GGTTCAAGAGGCCAATAATGTTGAAGCAGCACAGAAT CTGACCGTTCCGGGTAGCCTGCGTGCAGTTGATATTCC GGGACTGAAAGCCGATACCCCGTATCGTGTTAGCATTT ATGGTGTTATTCAGGGTTATCGTACCCCGATGCTGAGC ACCGATGTTAGCACAGCACGTGAACCGGAAATTGGTA ATCTGAATGTTAGTGATGTGACCCCGAAATCATTTAAT CTGAGCTGGACCGCAACCGATGGTATTTTTGATATGTT TACCATTGAAATTATTGATAGCAATCGCCTGCTGCAGA CCGCAGAACATAACATTAGCGGTGCAGAACGTACCGC ACATATTAGCGGTCTGCCTCCGAGCACCGATTTTATTG TTTATCTGAGCGGTATTGCACCGAGCATTCGTACCAAA ACCATTAGCACCACCGCAACCACCGAAGCACTGACCG CAATGGGTAGCCCGAAAGAAGTGATTTTTAGCGATATT ACCGAAAATAGCGCCACCGTTTCATGGCGTGCACCGAC CGCACAGGTTGAAAGCTTTCGTATTACCTATGTTCCGA TTACCGGTGGCACCCCGAGCATGGTTACCGTTGATGGC ACCAAAACCCAGACCCGTCTGGTTAAACTGATTCCGGG TGTTGAATATCTGGTTAGCATTATTGCCATGAAAGGCT TTGAAGAAAGCGAACCGGTTAGCGGTAGCTTTACCAC AGCTAGCGGCCTGAACGACATCTTCGAGGCTCAGAAA ATCGAATGGCACGAAGGTACCCATCACCATCACCACC ACTAA cynoTNC ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCT SEQ ID NO: 3 TGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAG AAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGG TGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTG GAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTT AAATTAACACAGTCTATGGCCATCATACGTTATATAGC TGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAG CGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGA TATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAG ACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTA CCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCA TAAAACATATTTAAATGGTGATCATGTAACCCATCCTG ACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACA TGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTT TGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGA TAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTT TGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCAT CCTCCAAAATCGGATGGTTCAACTAGTGGTTCTGGTCA TCACCATCACCATCACTCCGCGGGTCTGGTGCCACGCG GTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGCT AAATTCGAACGCCAGCACATGGACAGCCCAGATCTGG GTACCGGTGGTGGCTCCGGTATTGAGGGACGCGGGTCC ATGGGATATCGGGGATCCGAACTGGATACCCCGAAAG ATCTGCGTGTTAGCGAAACCGCAGAAACCAGCCTGAC CCTGTTTTGGAAAACACCGCTGGCAAAATTTGATCGTT ATCGTCTGAATTATAGCCTGCCGACCGGTCAGTGGGTT GGTGTTCAGCTGCCTCGTAATACCACCAGTTATGTTCT GCGTGGTCTGGAACCGGGTCAAGAATATAACGTTCTGC TGACCGCAGAAAAAGGTCGTCATAAAAGCAAACCGGC ACGTGTTAAAGCAAGCACCGAACAGGCACCGGAACTG GAAAATCTGACCGTTACCGAAGTTGGCTGGGATGGCCT GCGCCTGAACTGGACGGCTGCGGACCAGGCCTACGAA CACTTCGTTATCCAGGTGCAAGAAGCCAACAAAGTAG AAGCCGCTCAGAATCTGACGGTTCCGGGAAATCTGCGT GCAGTTGATATTCCGGGTCTGAAAGCAGCAACCCCGTA TACCGTTAGCATTTATGGTGTTATTCAGGGTTATCGTAC ACCGGTTCTGAGTGCCGAAGCCAGCACCGGTGAAACC CCGAATCTGGGTGAAGTTATGGTTAGCGAAGTGGGCTG GGATGCACTGAAACTGAATTGGACAGTTCCGGAAGGT GCCTATGAATACTTTTTCATTCAGGTTCAAGAAGCGGA TACCGTTGAAGCCGCTCAGAATCATACCGTTCCGGGTG GTCTGCGTAGCACCGATCTGCCTGGCCTGAAAGCCGCT ACCCATTACACCATTACCATTCGTGGTGTTACCCAGGA TTTTAGCACCACACCGCTGAGCGTTGAAGTTCTGACAG AAGAACTGCCGCAGCTGGGTGATCTGGCAGTTAGCGA AGTTGGTTGGGATGGTCTGCGTCTGAATTGGACCGCAG CAGATCAGGCATATGAACATTTTGTTATCCAGGTGCAA GAAGTGAACAAAGTTGAAGCAGCACAGAATCTGACCG TTCCGGGTAGCCTGCGTGCAGTTGATATTCCGGGTCTG AAAGCAGCAACCCCGTATACCGTTAGCATTTATGGTGT TATTCGCGGTTATCGTACACCGGTTCTGAGCGCAGAAG CAAGCACCGCAAAAGAACCGGAAATTGGTAATCTGAA CGTGAGCGATATTACACCGGAAAGTTTTAGCCTGAGCT GGACCGCAACCGATGGTATTTTTGAAACCTTTACCATC GAGATCATCGATAGCAATCGTCTGCTGGAAATCGTGGA ATATAACATTAGCGGTGCAGAACGTACCGCACATATTA GCGGTCTGCCTCCGAGCACCGATTTTATTGTTTATCTGA GCGGTCTGGCACCGAGCTTTCGTACCAAAACCATTAGC GCAACCGCAACCACCGAAGCACTGACCGCAATGGGTA GCCCGAAAGAAGTGATTTTTAGCGATATTACCGAAAAT AGCGCCACCGTTTCATGGCGTGCACCGACCGCACAGGT TGAAAGCTTTCGTATTACCTATGTTCCGATTACCGGTG GCACCCCGAGCATGGTTACCGTGGATGGCACCAAAAC CCAGACCCGTCTGGTTAAACTGGTTCCGGGTGTTGAAT ATCTGGTGAATATCATTGCCATGAAAGGCTTTGAAGAA AGCGAACCGGTTAGCGGTAGCTTTACCACCGCTAGCGG CCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGG CACGAAGGTACCCATCACCATCACCACCACTAA huTNC MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGD SEQ ID NO: 4 KWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKH NMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYD ALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSK YIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSA GLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIE GRGSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKF DRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYN VLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWD GLRLNWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLR AVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETPN LGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEADTV EAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTT PLSVEVLTEEVPDMGNLTVTEVSWDALRLNWTTPDGTY DQFTIQVQEADQVEEAHNLTVPGSLRSMEIPGLRAGTPYT VTLHGEVRGHSTRPLAVEVVTEDLPQLGDLAVSEVGWD GLRLNWTAADNAYEHFVIQVQEVNKVEAAQNLTLPGSL RAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTAKEPE IGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSNRLLETV EYNISGAERTAHISGLPPSTDFIVYLSGLAPSIRTKTISATA TTEALPLLENLTISDINPYGFTVSWMASENAFDSFLVTVV DSGKLLDPQEFTLSGTQRKLELRGLITGIGYEVMVSGFTQ GHQTKPLRAEIVTEAMGSPKEVIFSDITENSATVSWRAPT AQVESFRITYVPITGGTPSMVTVDGTKTQTRLVKLIPGVE YLVSIIAMKGFEESEPVSGSFTTASGLNDIFEAQKIEWHEG THHHHHH muTNC MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGD SEQ ID NO: 5 KWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKH NMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYD ALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSK YIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSA GLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIE GRGSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKF DRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYN VLLTAEKGRHKSKPARVKASTEEVPSLENLTVTEAGWDG LRLNWTADDLAYEYFVIQVQEANNVETAHNFTVPGNLR AADIPGLKVATSYRVSIYGVARGYRTPVLSAETSTGTTPN LGEVTVAEVGWDALTLNWTAPEGAYKNFFIQVLEADTT QTVQNLTVPGGLRSVDLPGLKAATRYYITLRGVTQDFGT APLSVEVLTEDLPQLGGLSVTEVSWDGLTLNWTTDDLAY KHFVVQVQEANNVEAAQNLTVPGSLRAVDIPGLKADTP YRVSIYGVIQGYRTPMLSTDVSTAREPEIGNLNVSDVTPK SFNLSWTATDGIFDMFTIEIIDSNRLLQTAEHNISGAERTA HISGLPPSTDFIVYLSGIAPSIRTKTISTTATTEALTAMGSPK EVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMV TVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFT TASGLNDIFEAQKIEWHEGTHHHHHH cynoTNC MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGD SEQ ID NO: 6 KWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKH NMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYD ALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSK YIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSA GLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIE GRGSMGYRGSELDTPKDLRVSETAETSLTLFWKTPLAKF DRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYN VLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWD GLRLNWTAADQAYEHFVIQVQEANKVEAAQNLTVPGNL RAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETP NLGEVMVSEVGWDALKLNWTVPEGAYEYFFIQVQEADT VEAAQNHTVPGGLRSTDLPGLKAATHYTITIRGVTQDFST TPLSVEVLTEELPQLGDLAVSEVGWDGLRLNWTAADQA YEHFVIQVQEVNKVEAAQNLTVPGSLRAVDIPGLKAATP YTVSIYGVIRGYRTPVLSAEASTAKEPEIGNLNVSDITPESF SLSWTATDGIFETFTIEIIDSNRLLEIVEYNISGAERTAHISG LPPSTDFIVYLSGLAPSFRTKTISATATTEALTAMGSPKEVI FSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVD GTKTQTRLVKLVPGVEYLVNIIAMKGFEESEPVSGSFTTA SGLNDIFEAQKIEWHEGTHHHHHH

TABLE 4 Sequences of TnC antigens used for affinity determination (reference Table 5) Antigen pETR # batch ID Protein Sequence SEQ ID NO GST huTNC MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 7 fn5 A1234 BC WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN fn6 B MLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDAL DVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYI AWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGL VPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGR GSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDR YRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLL TAEKGRHKSKPARVKASTEQAPELENLTVTEVGWDGLRL NWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLRAVDI PGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETPNLGEV VVAEVGWDALKLNWTAPEGAYEYFFIQVQEADTVEAAQ NLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVE VLTEEVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQ VQEADQVEEAHNLTVPGSLRSMEIPGLRAGTPYTVTLHGE VRGHSTRPLAVEVVTEDLPQLGDLAVSEVGWDGLRLNW TAADNAYEHFVIQVQEVNKVEAAQNLTLPGSLRAVDIPG LEAATPYRVSIYGVIRGYRTPVLSAEASTAKEPEIGNLNVS DITPESFNLSWMATDGIFETFTIEIIDSNRLLETVEYNISGAE RTAHISGLPPSTDFIVYLSGLAPSIRTKTISATATTEALPLLE NLTISDINPYGFTVSWMASENAFDSFLVTVVDSGKLLDPQ EFTLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKPLRA EIVTEAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITY VPITGGTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGF EESEPVSGSFTTASGLNDIFEAQKIEWHEGTHHHHHH GST MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 8 huTNCfn5 mu WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN A124 BC hu MLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV fn6 B DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDAL DVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYI AWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGL VPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGR GSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDR YRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLL TAEKGRHKSKPARVKASTEEVPSLENLTVTEAGWDGLRL NWTADDLAYEYFVIQVQEANNVETAHNFTVPGNLRAADI PGLKVATSYRVSIYGVARGYRTPVLSAETSTGTTPNLGEV TVAEVGWDALTLNWTAPEGAYKNFFIQVLEADTTQTVQ NLTVPGGLRSVDLPGLKAATRYYITLRGVTQDFGTAPLSV EVLTEDLPQLGGLSVTEVSWDGLTLNWTTDDLAYKHFVV QVQEANNVEAAQNLTVPGSLRAVDIPGLKADTPYRVSIY GVIQGYRTPMLSTDVSTAREPEIGNLNVSDVTPKSFNLSW TATDGIFDMFTIEIIDSNRLLQTAEHNISGAERTAHISGLPPS TDFIVYLSGIAPSIRTKTISTTATTEALTAMGSPKEVIFSDIT ENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKT QTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTASGLNDI FEAQKIEWHEGTHHHHHH GST TNC hu MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 9 fn5 B-C fn6 B WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN MLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDAL DVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYI AWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGL VPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGR GSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDR YRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLL TAEKGRHKSKPARVKASTAKEPEIGNLNVSDITPESFNLS WMATDGIFETFTIEIIDSNRLLETVEYNISGAERTAHISGLP PSTDFIVYLSGLAPSIRTKTISATATTEALPLLENLTISDINP YGFTVSWMASENAFDSFLVTVVDSGKLLDPQEFTLSGTQ RKLELRGLITGIGYEVMVSGFTQGHQTKPLRAEIVTAMGS PKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSM VTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSF TTASGLNDIFEAQKIEWHEGTHHHHHH GST huTNC MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 10 fn5 A1234 fn6 B WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN MLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDAL DVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYI AWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGL VPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGR GSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDR YRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLL TAEKGRHKSKPARVKASTEQAPELENLTVTEVGWDGLRL NWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLRAVDI PGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETPNLGEV VVAEVGWDALKLNWTAPEGAYEYFFIQVQEADTVEAAQ NLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVE VLTEEVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQ VQEADQVEEAHNLTVPGSLRSMEIPGLRAGTPYTVTLHGE VRGHSTRPLAVEVVTEDLPQLGDLAVSEVGWDGLRLNW TAADNAYEHFVIQVQEVNKVEAAQNLTLPGSLRAVDIPG LEAATPYRVSIYGVIRGYRTPVLSAEASTAKEAMGSPKEVI FSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVD GTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTAS GLNDIFEAQKIEWHEGTHHHHHH huTNC A4 B MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 11 WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN MLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDAL DVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYI AWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGL VPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGR GSMGYRGSEDLPQLGDLAVSEVGWDGLRLNWTAADNA YEHFVIQVQEVNKVEAAQNLTLPGSLRAVDIPGLEAATPY RVSIYGVIRGYRTPVLSAEASTASGLNDIFEAQKIEWHEGT HHHHHH huTNC A1 B MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 12 WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN MLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDAL DVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYI AWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGL VPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGR GSMGYRGSEQAPELENLTVTEVGWDGLRLNWTAADQAY EHFIIQVQEANKVEAARNLTVPGSLRAVDIPGLKAATPYT VSIYGVIQGYRTPVLSAEASTASGLNDIFEAQKIEWHEGTH HHHHH

Example 2

Selection of Anti-TnC Antibodies from Generic Fab Libraries

Anti-TnC antibodies were selected from two different generic phage display libraries: DP88-4 (clone 18D4) and lambda-DP47 (clone 11C7).

Library Construction

The DP88-4 library was constructed on the basis of human germline genes using the V-domain pairing Vk1_5 (kappa light chain) and VH1_69 (heavy chain) comprising randomized sequence space in CDR3 of the light chain (L3, 3 different lengths) and CDR3 of the heavy chain (H3, 3 different lengths). Library generation was performed by assembly of 3 PCR-amplified fragments applying splicing by overlapping extension (SOE) PCR. Fragment 1 comprises the 5′ end of the antibody gene including randomized L3, fragment 2 is a central constant fragment spanning from L3 to H3 whereas fragment 3 comprises randomized H3 and the 3′ portion of the antibody gene. The following primer combinations were used to generate these library fragments for DP88-4 library: fragment 1 (forward primer LMB3 combined with reverse primers Vk1_5_L3r_S or Vk1_5_L3r_SY or Vk1_5_L3r_SPY), fragment 2 (forward primer RJH31 combined with reverse primer RJH32) and fragment 3 (forward primers DP88-v4-4 or DP88-v4-6 or DP88-v4-8 combined with reverse primer fdseqlong), respectively. PCR parameters for production of library fragments were 5 min initial denaturation at 94° C., 25 cycles of 1 min 94° C., 1 min 58° C., 1 min 72° C. and terminal elongation for 10 min at 72° C. For assembly PCR, using equimolar ratios of the gel-purified single fragments as template, parameters were 3 min initial denaturation at 94° C. and 5 cycles of 30 s 94° C., 1 min 58° C., 2 min 72° C. At this stage, outer primers (LMB3 and fdseqlong) were added and additional 20 cycles were performed prior to a terminal elongation for 10 min at 72° C. After assembly of sufficient amounts of full length randomized Fab constructs, they were digested NcoI/NheI and ligated into similarly treated acceptor phagemid vector. Purified ligations were used for ˜60 transformations into electrocompetent E. coli TG1. Phagemid particles displaying the Fab library were rescued and purified by PEG/NaCl purification to be used for selections. These library construction steps were repeated three times to obtain a final library size of 4.4×10⁹. Percentages of functional clones, as determined by C-terminal tag detection in dot blot, were 92.6% for the light chain and 93.7% for the heavy chain, respectively.

The lambda-DP47 library was constructed on the basis of human germline genes using the following V-domain pairings: V13_19 lambda light chain with VH3_23 heavy chain. The library was randomized in CDR3 of the light chain (L3) and CDR3 of the heavy chain (H3) and was assembled from 3 fragments by “splicing by overlapping extension” (SOE) PCR. Fragment 1 comprises the 5′ end of the antibody gene including randomized L3, fragment 2 is a central constant fragment spanning from the end of L3 to the beginning of H3 whereas fragment 3 comprises randomized H3 and the 3′ portion of the Fab fragment. The following primer combinations were used to generate library fragments for library: fragment 1 (LMB3-V1_3_19_L3r_V/V1_3_19_L3r_HV/V1_3_19_L3r_HLV), fragment 2 (RJH80-DP47CDR3_ba (mod)) and fragment 3 (DP47-v4-4/DP47-v4-6/DP47-v4-8-fdseqlong). PCR parameters for production of library fragments were 5 min initial denaturation at 94° C., 25 cycles of 60 sec 94° C., 60 sec 55° C., 60 sec 72° C. and terminal elongation for 10 min at 72° C. For assembly PCR, using equimolar ratios of the 3 fragments as template, parameters were 3 min initial denaturation at 94° C. and 5 cycles of 60 s 94° C., 60 sec 55° C., 120 sec 72° C. At this stage, outer primers were added and additional 20 cycles were performed prior to a terminal elongation for 10 min at 72° C. After assembly of sufficient amounts of full length randomized Fab fragments, they were digested with NcoI/NheI alongside with similarly treated acceptor phagemid vector. 15 μg of Fab library insert were ligated with 13.3 μg of phagemid vector. Purified ligations were used for 60 transformations resulting in 1.5×10⁹ transformants Phagemid particles displaying the Fab library were rescued and purified by PEG/NaCl purification to be used for selections.

Phage Display Selections & ELISA Screening

Human GST-fused TnC fn5 A1234 BC fn6 as antigen for the phage display selections was expressed in E. coli and in vivo site-specifically biotinylated via co-expression of BirA biotin ligase at the avi-tag recognition sequence located at the C-terminus of the fusion protein (production of antigens according to Example 1, sequences derived from Table 4). This antigen comprises the human TnC extra splice domains A1, A2, A3, A4, B, and C, located between the two fibronectin type III domains 5 and 6. The phage display selections aimed at selecting binders to any of these extra splice domains and determine the domain specificity in a subsequent step by surface plasmon resonance using additional antigen constructs comprising fewer extra splice domains.

Selection rounds (biopanning) were performed in solution according to the following pattern: 1. Pre-clearing of ˜10¹² phagemid particles with an unrelated GST-fusion protein that also carried an avi-tag and His6-tag similar to the TnC target cell antigen to deplete the libraries of antibodies recognizing the three different tags, 2. incubation of the pre-cleared phagemid particles in the supernatant with 100 nM biotinylated human GST-fused TnC fn5 A1234 BC fn6 for 0.5 hours in the presence of an unrelated non-biotinylated GST-fusion protein for further depletion of tag-binders in a total volume of 1 ml, 3. capture of biotinylated human GST-fused TnC fn5 A1234 BC fn6 and attached specifically binding phage by transfer to 4 wells of a neutravidin pre-coated microtiter plate for 10 minutes (in rounds 1 & 3), 4. washing of respective wells using 5×PBS/Tween20 and 5×PBS, 5. elution of phage particles by addition of 250 μl 100 mM TEA (triethylamine) per well for 10 minutes and neutralization by addition of 500 μl 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells, 6. re-infection of log-phase E. coli TG1 cells with the supernatant of eluted phage particles, infection with helperphage VCSM13, incubation on a shaker at 30° C. over night and subsequent PEG/NaCl precipitation of phagemid particles to be used in the next selection round. Selections were carried out over 3 rounds using constant antigen concentrations of 100 nM. In round 2, in order to avoid enrichment of binders to neutravidin, capture of antigen: phage complexes was performed by addition of 5.4×10⁷ streptavidin-coated magnetic beads. Specific binders were identified by ELISA after rounds 2 and 3 as follows: 100 μl of 100 nM biotinylated human GST-fused TnC fn5 A1234 BC fn6 were coated on neutravidin plates. Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags using an anti-Flag/HRP secondary antibody. Clones exhibiting signals on human GST-fused TnC fn5 A1234 BC fn6 and being negative on an unrelated GST-fusion protein carrying the same tags as the target, were short-listed for further analyses. They were bacterially expressed in a 0.5 liter culture volume, affinity purified and further characterized by SPR-analysis using BioRad's ProteOn XPR36 biosensor to test cross-reactivity to murine TnC and to determine which extra splice domains the antibodies recognize.

SPR-Analysis Using BioRad's ProteOn XPR36 Biosensor

Affinities (K_(D)) of selected clones were measured by surface plasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. with biotinylated human and murine TnC antigens immobilized on NLC chips by neutravidin capture. Immobilization of antigens (ligand): Recombinant antigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute in vertical orientation. Injection of analytes: For ‘one-shot kinetics’ measurements, injection direction was changed to horizontal orientation, two-fold dilution series of purified Fab were injected simultaneously along separate channels 1-5, with association times of 200 s, and dissociation times of 240 s, respectively. Buffer (PBST) was injected along the sixth channel to provide an “in-line” blank for referencing. Association rate constants (k_(on)) and dissociation rate constants (k_(off)) were calculated using a simple one-to-one Langmuir binding model in ProteOn Manager v3.1 software by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K_(D)) was calculated as the ratio k_(off)/k_(on). Table 5 lists the equilibrium dissociation constants (K_(D)) of the two selected clones 18D4 and 11C7 for several TnC antigens differing in species and composition of the extra splice domains.

TABLE 5 Equilibrium dissociation constants (K_(D)) for clones 18D4 and 11C7 clone 18D4 clone 11C7 antigen K_(D) [nM] K_(D) [nM] GST huTNC fn5 A1234 BC fn6 B 4.0 2.3 GST huTNCfn5 mu A124 BC hu fn6 B 11.2 1.9 GST TNC hu fn5 B-C fn6 B n.a. 1.0 GST huTNC fn5 A1234 fn6 B 5.0 n.a. huTNC A4 B 2.0 n.a. huTNC A1 B 5.6 n.a.

TABLE 6 DNA sequence of generic phage-displayed DP88-4 library (Vk1_5/VH1_69) template used for PCRs Construct Base pair sequence SEQ ID NO pRJH33 library ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTAT SEQ ID NO: 13 template DP88-4 TACTCGCGGCCCAGCCGGCCATGGCCGACATCCAGATGAC library; complete CCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACCGTG Fab coding TCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTG region GTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA comprising PelB GCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTC leader sequence + CCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCA Vk1_5 kappa CTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACT V-domain + CL TATTACTGCCAACAGTATAATAGTTATTCTACGTTTGGCCA constant domain GGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACC for light chain ATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT and PelB + CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT VH1_69 V- CCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC domain + CH1 CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG constant domain GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG for heavy chain) ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA CAAAGAGCTTCAACAGGGGAGAGTGTGGAGCCGCAGAAC AAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAG ACTACAAGGACGACGACGACAAGGGTGCCGCATAATAAG GCGCGCCAATTCTATTTCAAGGAGACAGTCATATGAAATA CCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTG CCCAGCCGGCGATGGCCCAGGTGCAATTGGTGCAGTCTGG GGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC TGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAA GCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGA TGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGC ACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAA ATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAG ATCTGAGGACACCGCCGTGTATTACTGTGCGAGACTATCC CCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGA CCACCGTGACCGTCTCCTCAGCTAGCACCAAAGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA CAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCC CAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCAC CATCACCATCACGCCGCGGCA

TABLE 7 Base pair sequence of DP88-4 library (Vk1_5/VH1_69) germline template Construct Base pair sequence SEQ ID NO Fab light chain GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATC SEQ ID NO: 14 V1_5 TGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAG AGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTT GGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCC GGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTG ATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTAT TCTACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTA CGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAA GGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGT GTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAA CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTG GAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGA ATGGAGCCGCAGACTACAAGGACGACGACGACAAGGGTG CCGCA Fab heavy chain CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG SEQ ID NO: 15 VH1_69 CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG GCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGC CCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCT ATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCA GGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTA CATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGT GTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTT ATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCT CAGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACC CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC AGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAA AGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGC CGCAAGCACTAGTGCCCATCACCATCACCATCACGCCGCG GCA

TABLE 8 Amino acid sequence of DP88-4 library (Vk1_5/VH1_69) germline template Construct Base pair sequence SEQ ID NO Fab light chain DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGK SEQ ID NO: 16 Vk1_5 APKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATY YCQQYNSYSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECGAAEQKLISEEDLNGAADYKDDDDKGAA Fab heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP SEQ ID NO: 17 VH1_69 (DP88) GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMEL SSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDAAASTSAHHHHHHAAA

TABLE 9 Primer sequences used for generation of DP88-4 library (Vk1_5/VH1_69) Primer name Primer sequence 5′-3′ SEQ ID NO LMB3 CAGGAAACAGCTATGACCATGATTAC SEQ ID NO: 18 Vk1_5_L3r_S CTCGACTTTGGTGCCCTGGCCAAACGTS BA A TA SEQ ID NO: 19 C GA A TT A TACTGTTGGCAGTAATAAGTTGCAAAATCAT Vk1_5_L3r_SY CTCGACTTTGGTGCCCTGGCCAAACGTM HR S GR A TA C GA SEQ ID NO: 20 A TT A TACTGTTGGCAGTAATAAGTTGCAAAATCAT Vk1_5_L3r_SPY CTCGACTTTGGTGCCCTGGCCAAACGTM HH M SS S GR A TA C SEQ ID NO: 21 GA A TT A TACTGTTGGCAGTAATAAGTTGCAAAATCAT RJH31 ACGTTTGGCCAGGGCACCAAAGTCGAG SEQ ID NO: 22 RJH32 TCTCGCACAGTAATACACGGCGGTGTCC SEQ ID NO: 23 DP88-v4-4 GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-3-4-GAC- SEQ ID NO: 24 TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC DP88-v4-6 GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-2-2-3-4- SEQ ID NO: 25 GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC DP88-v4-8 GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-2-2-2-2-3-4- SEQ ID NO: 26 GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC fdseqlong GACGTTAGTAAATGAATTTTCTGTATGAGG SEQ ID NO: 27 Underlined bases: 60% given sequence and 40% N; Bases in italics: 60% given sequence and 40% M. 1: G/D = 20%, E/V/S = 10%, A/P/R/L/T/Y = 5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E = 4.6%; 3: G/A/Y = 20%, P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%.

TABLE 10 DNA sequence of generic phage-displayed lambda-DP47 library (Vl3_19/VH3_23) template used for PCRs Construct Base pair sequence SEQ ID NO pRJH53 library ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTAT SEQ ID NO: 28 template of TACTCGCGGCCCAGCCGGCCATGGCCTCGTCTGAGCTGAC lambda-DP47 TCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTC library AGGATCACATGCCAAGGAGACAGCCTCAGAAGTTATTATG Vl3_19/VH3_23; CAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTAC complete Fab TTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCC coding region AGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCC comprising PelB TTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACT leader sequence + ATTACTGTAACTCCCGTGATAGTAGCGGTAATCATGTGGT Vl3_19 lambda ATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGACAACC V-domain + CL CAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGC constant domain GAGGAATTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGA for light chain TCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAA and PelB + GGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCAC VH3_23 V- CACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAG domain + CH1 CAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCA constant domain CAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCAC for heavy chain CGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGCGGAGC including tags CGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGG AGCCGCAGACTACAAGGACGACGACGACAAGGGTGCCGC ATAATAAGGCGCGCCAATTCTATTTCAAGGAGACAGTCAT ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGC TCCTCGCTGCCCAGCCGGCGATGGCCGAGGTGCAATTGCT GGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG AGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTA TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACA TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACA GCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAA ACCGTTTCCGTATTTTGACTACTGGGGCCAAGGAACCCTG GTCACCGTCTCGAGTGCTAGCACCAAAGGCCCATCGGTCT TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAC AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC CCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAAT CTTGTGACGCGGCCGCAAGCACTAGTGCCCATCACCATCA CCATCACGCCGCGGCA

TABLE 11 Base pair sequence of lambda-DP47 library (Vl3_19/VH3_23) germline template Construct Base pair sequence SEQ ID NO Fab light chain TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTT SEQ ID NO: 29 Vl3_19 GGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCT CAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGG ACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGG CCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAG GAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGA AGATGAGGCTGACTATTACTGTAACTCCCGTGATAGTAGC GGTAATCATGTGGTATTCGGCGGAGGGACCAAGCTGACCG TCCTAGGACAACCCAAGGCTGCCCCCAGCGTGACCCTGTT CCCCCCCAGCAGCGAGGAATTGCAGGCCAACAAGGCCAC CCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTG ACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCC GGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAAC AAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGC AGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCC ACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCG AGTGCAGCGGAGCCGCAGAACAAAAACTCATCTCAGAAG AGGATCTGAATGGAGCCGCAGACTACAAGGACGACGACG ACAAGGGTGCCGCA Fab heavy chain GAGGTGCAATTGCTGGAGTCTGGGGGAGGCTTGGTACAGC SEQ ID NO: 30 VH3_23 CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATT CACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTA GTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCG GTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT CTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTA TATTACTGTGCGAAACCGTTTCCGTATTTTGACTACTGGGG CCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAA GGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCA CCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGT GCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC GTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAA GTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTG CCCATCACCATCACCATCACGCCGCGGCA

TABLE 12 Amino acid sequence of lambda-DP47 library (Vl3_19/VH3_23) germline template Construct Base pair sequence SEQ ID NO Fab light chain SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQ SEQ ID NO: 31 Vl3_19 APVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEAD YYCNSRDSSGNHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEE LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECSGAAEQKLISEEDLNGAADYKDDDDKGAA Fab heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP SEQ ID NO: 32 VH3_23 (DP47) GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDAAASTSAHHHHHHAAA

TABLE 13 Primer sequences used for generation of lambda-DP47 library (Vl3_19/VH3_23) Primer name Primer sequence 5′-3′ SEQ ID NO LMB3 See Table 9 SEQ ID NO: 18 Vl_3_19_L3r_V GGACGGTCAGCTTGGTCCCTCCGCCGAATAC V

A

SEQ ID NO: 33 A

G

A

 A

A

GGAGTTACAGTAATAGTCAGCCTCATCTTCCGC underlined: 60% original base and 40% randomization as M bold and italic: 60% original base and 40% randomization as N Vl_3_19_L3r_H V GGACGGTCAGCTTGGTCCCTCCGCCGAATAC C

A

SEQ ID NO: 34 A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTCCGC underlined: 60% original base and 40% randomization as M bolded and italic: 60% original base and 40% randomization as N Vl_3_19_L3r_H GGACGGTCAGCTTGGTCCCTCCGCCGAATAC R

V

SEQ ID NO: 35 LV A

 A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTC CGC underlined: 60% original base and 40% randomization as M bolded and italic: 60% original base and 40% randomization as N RJH80 TTCGGCGGAGGGACCAAGCTGACCGTCC SEQ ID NO: 36 DP47CDR3_ba CGCACAGTAATATACGGCCGTGTCC SEQ ID NO: 37 (mod.) DP47-v4-4 CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-3-4-GAC- SEQ ID NO: 38 TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG DP47-v4-6 CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-3-4- SEQ ID NO: 39 GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG DP47-v4-8 CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-2-2-3- SEQ ID NO: 40 4-GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG fdseqlong See Table 9 SEQ ID NO: 27 Trinucleotide mixtures in randomized primers: 1 (G/D = 20%, E/V/S = 10%, A/P/R/L/T/Y = 5%); 2 (G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E = 4.6%); 3 (G/A/Y = 20, P/W/S/D/T = 8%); 4 (F = 46%, L/M = 15%, G/I/Y = 8%); 5 (K = 70%, R = 30%)

Example 3 Cloning of Variable Antibody Domains into Expression Vectors

The variable regions of heavy and light chain DNA sequences of the selected anti-TnC binders (Table 14 to Table 19) were subcloned in frame with either the constant heavy chain or the constant light chain of human IgG1. The antibodies have been prepared either as wild type human IgG1 backbone or as variant containing Pro329Gly, Leu234Ala and Leu235Ala mutations, which have been introduced to abrogate binding to Fc gamma receptors according to the method described in International Patent Appl. Publ. No. WO 2012/130831 A1.

The CDR sequences of the anti-TnC binder are shown in Table 16 to Table 19. The base pair and amino acid sequences of the anti-TnC IgGs are shown in Table 20 and Table 21. The base pair and amino acid sequences of the anti-TnC P319GLALA IgGs are shown in Table 22 and Table 23. All antibody-encoding sequences were cloned into an expression vector, which drives transcription of the insert with a chimeric MPSV promoter and contains a synthetic polyA signal sequence located at the 3′ end of the CDS. In addition, the vector contains an EBV OriP sequence for episomal maintenance of the plasmid.

LCDR3 of clone 11C7 (NSINSTRNEV (SEQ ID NO: 60)), as selected by phage display, contains a potential N-glycosylation site, i.e. NST, which can potentially be removed by amino acid substitutions to facilitate production of a homogeneous product. At the same time, binding to the target should be retained. N (position 1) could preferentially be substituted by Q, S or T. Alternatively, S (position 2) could be replaced by P. Alternatively, T (position 3) could be substituted preferentially by G or N or by any other proteinogenic amino acid except for S or C. Whichever substitution(s) would be the best, can be determined empirically by those skilled in the art.

TABLE 14 Variable region base pair sequences for phage-derived anti-TnC antibodies Clone Chain Base pair sequence SEQ ID NO 18D4 VL GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCAT SEQ ID NO: 41 CTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCA GAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACC AGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGT TTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGAT CCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCC TGATGATTTTGCAACTTATTACTGCCAACAGAATAAGAAG TTTCCTTCGGGGACGTTTGGCCAGGGCACCAAAGTCGAGA TCAAG VH CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG SEQ ID NO: 42 CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG GCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG CCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGG CAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGC CTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGC CGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTG GACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 11C7 VL TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCT SEQ ID NO: 43 TGGGACAGACAGTCAGGGTCACATGCCAAGGAGACAGCC TCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAG GACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCG GCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCA GGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCG GAAGATGAGGCTGACTATTACTGTAACTCCATTAATAGTA CTCGTAATGAGGTATTCGGCGGAGGGACCAAGCTGACCG TCCTA VH GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAG SEQ ID NO: 44 CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGAT TCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGCGG TAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAAAGACAATTCCAAGAACACGCTG TATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCC GTATATTACTGTGCGAAAACTTCTCCGCGTGTTCCGCTGG ACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT

TABLE 15 Variable region polypeptide sequences for phage-derived anti-TnC antibodies Clone Chain Polypeptide sequence 18D4 VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGK SEQ ID NO: 45 APKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATY YCQQNKKFPSGTFGQGTKVEIK VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP SEQ ID NO: 46 GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME LSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTVTVSS 11C7 VL SSELTQDPAVSVALGQTVRVTCQGDSLRSYYASWYQQKPG SEQ ID NO: 47 QAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEA DYYCNSINSTRNEVFGGGTKLTVL VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP SEQ ID NO: 48 GKGLEWVSAISGSGGSTYYADSVKGRFTISKDNSKNTLYLQ MNSLRAEDTAVYYCAKTSPRVPLDYWGQGTLVTVSS

TABLE 16 CDR base pairs sequences of the anti-TnC antibody light chains SEQ ID SEQ ID SEQ ID clone NO LCDR1 NO LCDR2 NO LCDR3 18D4 SEQ ID CGTGCCAGTCA SEQ ID GATGCCTCCA SEQ ID CAACAGAATAAG NO: 49 GAGTATTAGTA NO: 50 GTTTGGAAAGT NO: 51 AAGTTTCCTTCGG GCTGGTTGGCC GGACG 11C7 SEQ ID CAAGGAGACA SEQ ID GGTAAAAACA SEQ ID AACTCCATTAATA NO: 52 GCCTCAGAAGT NO: 53 ACCGGCCCTCA NO: 54 GTACTCGTAATGA TATTATGCAAGC GGTA

TABLE 17 CDR polypeptides sequences of the anti-TnC antibody light chains SEQ ID SEQ ID SEQ ID clone NO LCDR1 NO LCDR2 NO LCDR3 18D4 SEQ ID RASQSISSWLA SEQ ID DASSLES SEQ ID QQNKKFPSGT NO: 55 NO: 56 NO: 57 11C7 SEQ ID QGDSLRSYYAS SEQ ID GKNNRPS SEQ ID NSINSTRNEV NO: 58 NO: 59 NO: 60

TABLE 18 CDR base pairs sequences of the anti-TnC antibody heavy chains SEQ ID SEQ ID SEQ ID clone NO HCDR1 NO HCDR2 NO HCDR3 18D4 SEQ ID AGCTACGCTAT SEQ ID GGGATCATCC SEQ ID GGTAACTTCTACGG NO: 61 AAGC NO: 62 CTATCTTTGG NO: 63 TGGTCTGGACTAC TACAGCAAAC TACGCACAGA AGTTCCAGGGC 11C7 SEQ ID GGATTCACCTT SEQ ID GCTATTAGCG SEQ ID ACTTCTCCGCGTGT NO: 64 TAGCAGTTATG NO: 65 GTAGTGGTGG NO: 66 TCCGCTGGACTAC CCATGAGC TAGCACATAC TACGCAGACT CCGTGAAGGGC

TABLE 19 CDR polypeptide sequences of the anti-TnC antibody heavy chains SEQ ID SEQ ID SEQ ID clone NO HCDR1 NO HCDR2 NO HCDR3 18D4 SEQ ID SYAIS SEQ ID GIIPIFGTANYA SEQ ID GNFYGGLDY NO: 67 NO: 68 QKFQG NO: 69 11C7 SEQ ID GFTFSSYAMS SEQ ID AISGSGGSTYY SEQ ID TSPRVPLDY NO: 70 NO: 71 ADSVKG NO: 72

TABLE 20 Base pair sequences of anti-TnC clones in wild type human IgG1 format Clone Chain Base pair sequence SEQ ID NO 18D4 Light GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATC SEQ ID NO: 73 chain TGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGA GTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGG AAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGA AAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGA CAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGAT TTTGCAACTTATTACTGCCAACAGAATAAGAAGTTTCCTTC GGGGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGT ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCT GAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCG CCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT Heavy CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGC SEQ ID NO: 74 chain CTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGC ACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCC TGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCT TTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGT CACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGG AGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTAC TGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGG CCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGG GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTC CAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCC CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAG CACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAG CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC CCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 11C7 Light TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTT SEQ ID NO: 75 chain GGGACAGACAGTCAGGGTCACATGCCAAGGAGACAGCCTC AGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGAC AGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCC TCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAA CACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATG AGGCTGACTATTACTGTAACTCCATTAATAGTACTCGTAAT GAGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTC AACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGC AGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCC TGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGG AAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCA CCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAG CAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCAC AGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCG TGGAGAAAACCGTGGCCCCCACCGAGTGCAGC Heavy GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC SEQ ID NO: 76 chain TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCA CCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGCGGTAGTG GTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTC ACCATCTCCAAAGACAATTCCAAGAACACGCTGTATCTGCA GATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTAC TGTGCGAAAACTTCTCCGCGTGTTCCGCTGGACTACTGGGG CCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGG GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTC CAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCC CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAG CACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAG CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC CCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

TABLE 21 Polypeptide sequences of anti-TnC clones in wild type human IgG1 format Clone Chain Polypeptide sequence SEQ ID NO 18D4 Light DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGK SEQ ID NO: 77 chain APKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATY YCQQNKKFPSGTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP SEQ ID NO: 78 chain GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMEL SSLRSEDTAVYYCAKGNFYGGLDYWGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11C7 Light SSELTQDPAVSVALGQTVRVTCQGDSLRSYYASWYQQKPGQ SEQ ID NO: 79 chain APVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEAD YYCNSINSTRNEVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP SEQ ID NO: 80 chain GKGLEWVSAISGSGGSTYYADSVKGRFTISKDNSKNTLYLQ MNSLRAEDTAVYYCAKTSPRVPLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE 22 Base pair sequences of anti-TnC clones in P329GLALA human IgG1 format Clone Chain Base pair sequence 18D4 Light See Table 20 SEQ ID NO: 73 chain Heavy CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG SEQ ID NO: 81 chain CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG PGLALA GCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG CCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGG CAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGC CTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGC CGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTG GACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC AGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCA GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACC TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAA 11C7 Light See Table 20 SEQ ID NO: 75 chain Heavy GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAG SEQ ID NO: 82 chain CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGAT PGLALA TCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGC TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGCGG TAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGG CCGGTTCACCATCTCCAAAGACAATTCCAAGAACACGCTG TATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCC GTATATTACTGTGCGAAAACTTCTCCGCGTGTTCCGCTGG ACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTG CTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAG GTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCC GGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA ACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

TABLE 23 Polypeptide sequences of anti-TnC clones in P329GLALA human IgG1 format Clone Chain Polypeptide sequence 18D4 Light See Table 21 SEQ ID NO: 77 chain Heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP SEQ ID NO: 83 chain GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME LSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11C7 Light See Table 21 SEQ ID NO: 79 chain Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP SEQ ID NO: 84 chain GKGLEWVSAISGSGGSTYYADSVKGRFTISKDNSKNTLYLQ MNSLRAEDTAVYYCAKTSPRVPLDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 4

Purification of Anti-TnC IgGs

All genes were transiently expressed under control of a chimeric MPSV promoter consisting of the MPSV core promoter combined with the CMV promoter enhancer fragment. The expression vector also contains the oriP region for episomal replication in EBNA (Epstein Barr Virus Nuclear Antigen) containing host cells.

The anti-TnC IgGs were produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:1 ratio (“vector HC”:“vector HLC”).

For a 200 mL production in 500 mL shake flasks, 250 million HEK293 EBNA cells were seeded 24 hours before transfection in Excell media with supplements. For transfection, the cells were centrifuged for 5 minutes at 210×g, and supernatant was replaced by pre-warmed CD-CHO medium. Expression vectors were mixed in 20 mL CD-CHO medium to a final amount of 200 μg DNA. After addition of 540 μL PEI (1 mg/mL), the solution was vortexed for 15 seconds and incubated for 10 minutes at room temperature. Afterwards, cells were mixed with the DNA/PEI solution, transferred to a 500 mL shake flask and incubated for 3 hours at 37° C. in an incubator with a 5% CO₂ atmosphere and shaking at 165 rpm. After the incubation, 160 mL Excell medium with supplements was added and cells were cultured for 24 hours. At this point the valproic acid concentration is 1 mM (the media comprises additionally g/L PepSoy and 6 mM L-Glutamine) 24 hours after transfection the cells are supplement with an amino acid and glucose feed at 12% final volume (24 mL) and 3 g/L glucose (1.2 mL from 500 g/L stock). After culturing for 7 days, the cell supernatant was collected by centrifugation for 45 minutes at 2000-3000×g. The solution was sterile filtered (0.22 μm filter), supplemented with sodium azide to a final concentration of 0.01% (w/v), and kept at 4° C.

Purification of anti-TnC IgGs from cell culture supernatants was carried out by affinity chromatography using MabSelectSure. The protein was concentrated and filtered prior to loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM NaCl, 0.01% Tween-20 solution of pH 6.0.

For affinity chromatography, the supernatant was loaded on a ProtA MabSelect Sure column (CV=6 mL, GE Healthcare) equilibrated with 36 mL 20 mM Sodium Citrate, 20 mM Sodium Phosphate, pH 7.5. Unbound protein was removed by washing with 6-10 column volumes of a buffer containing 20 mM Sodium Citrate, 20 mM Sodium Phosphate, pH 7.5. The bound protein was eluted using a linear pH-gradient of 15 CVs of sodium chloride (from 20 to 100%) of 20 mM Sodium Citrate, 100 mM Sodium Chloride, 100 mM Glycine, 0.01% (v/v) Tween-20, pH 3.0. The column was then washed with 4 column volumes of a solution containing 20 mM Sodium Citrate, 100 mM Sodium Chloride, 100 mM Glycine, 0.01% (v/v) Tween-20, pH 3.0 followed by a re-equilibration step.

The pH of the collected fractions was adjusted by adding 1/10 (v/v) of 0.5 M Na₂HPO₄, pH 8.0. The protein was concentrated and filtered prior to loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM NaCl, pH 6.0, 0.01% Tween20.

The protein concentration of purified IgGs was determined by measuring the OD at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

Purity and molecular weight of the IgGs were analyzed by CE-SDS in the presence and absence of a reducing agent (Invitrogen, USA) using a LabChipGXII (Caliper). The aggregate content of the purified protein was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in a 25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C. (Table 24).

TABLE 24 Biochemical analysis of anti-TnC IgG1 Yield Monomer Clone [mg/l] [%] a-TnC(18D4) 6.8 98.3 huIgG1 a-TnC(18D4) 26.7 100 P329GLALA huIgG1

Example 5

Surface Plasmon Resonance (TnC Binding)

Binding of the anti-TnC IgGs 18D4, 11C7 and the Fab fragment of anti-TnC 18D4 to human, murine and cynomolgus TnC (antigens according to Table 4) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

A 20 μg/ml stock solution of biotinylated human, murine or cynomolgus TnC was injected on a SA chip, with the aim to immobilize up to 100 RU using the immobilization wizard function. Final immobilization levels were between 79-400 RU.

The anti-TnC IgGs 18D4 (PGLALA and wtFc), 11C7 (rb IgG) and 18D4 Fab fragment were then immediately passed over the chip surface at a concentration ranging from 0.02-12.5 nM (IgGs) and 0.02-50 nM (Fab fragment) with a flow rate of 30 μl/min for 180 s followed by a dissociation step of 180s. An additional dissociation step of 1800s was performed for the highest concentration of IgG. After each step the surface was regenerated following two sequential injections of 10 mM glycine pH2.1 for 60 s. Bulk refractive index differences were corrected for by subtracting the response obtained in a reference flow cell without TNC. Affinity and avidity were calculated using the Langmuir 1:1 kinetic model. However, as the K_(D) for the 1:1 fitting of bivalent interactions is an apparent value only, it should only be used for comparisons.

TABLE 26 KD values and species cross-reactivity hu TNC cyno TNC mu TNC Construct KD (nM) KD (nM) KD (nM) 18D4 IgG wt Fc 0.093 0.036 0.061 18D4 IgG PG/LALA 0.032 0.011 0.030 llC7 rb IgG 0.029 nd nd 18D4 Fab fragment 1.44 6.69 6.5

The anti-TnC binder 18D4 cross-reacts with mouse and cynomolgus TnC with similar avidity in the pM range. Monovalent binding of the 18D4 Fab fragment to TnC results in a K_(D) in the low nM range. Anti-TNC binder 11C7 also binds human TnC in the pM range. Species cross-reactivity could not be assessed for binder 11C7 due to absence of the C domain in murine and cynomolgus TNC constructs used in this Biacore experiment (Table 26).

Thermal Stability

The thermal stability was monitored by Static Light Scattering (SLS) and by measuring the intrinsic protein fluorescence in response to applied temperature stress. 30 μg of filtered protein sample with a protein concentration of 1 mg/ml was applied in duplicate to an Optim 2 instrument (Avacta Analytical Ltd). The temperature was ramped from 25 to 85° C. at 0.1° C./min, with the radius and total scattering intensity being collected. For determination of intrinsic protein fluorescence the sample was excited at 266 nm and emission was collected between 275 nm and 460 nm. Both IgGs have an aggregation temperature of 62° C. (Table 26).

TABLE 26 Thermal stability Construct Tagg (° C.) 18D4 IgG PGLALA 62 18D4 IgG wtFc 62

Example 6 Results of TnC Staining in LS174T Xenograft Derived Tumor Tissue and Human Tissue Array

Anti-TnC clones 18D4 and 11C7 as rabbit IgG antibodies were tested in LS174T xenograft derived tumor tissue and human tissue array for detection of TnC with an immunohistochemistry technique.

The LS174T colorectal carcinoma frozen tumor samples were sectioned in a Cryostat at 12 μm thickness, mounted on superfrost slides (Thermo Scientific, Germany). Frozen samples of human tissue array including paired normal and tumor samples were purchased from Biochain (San Francisco, USA). Tissue sections were allowed to thaw for 30 minutes. Slides were washed in PBS, fixed in cold acetone for 10 minutes and an incubation step with 0.03% hydrogen peroxidase in water was performed to block the endogeneous peroxidase. The sections were then incubated for 1 h with 5% goat serum in PBS followed by overnight incubation with 0.5 μg/ml anti-TnC clone 18D4 or 11C7 or an isotype control antibody (Serotec, Germany) at 4° C. Afterwards, the sections were washed with PBS three times for 5 minutes each and developed using the peroxidase Rabbit Vectastain ABC kit following the manufacturers' instructions (PK-6101, Vector laboratories, California, USA). Slides were then dehydrated in increasing concentration of ethanol and incubated 2 minutes in Xylene. One drop of Permount mounting medium (Fischer Chemical, Germany) was added to the sections and coverslip. Images were obtained with Olympus scanner VS120 (Olympus, Germany) and analyzed.

The pattern of histological staining in LS174T xenografts tumors corresponds to specific TnC stroma fibers (FIGS. 1A-1C). The TnC staining, for both clones 18D4 and 11C7, is overall expressed with moderate intensity. Negative isotype control signal validates the specificity of the technique. Specificity of the staining is validated by negative isotype control signals in the corresponding histological staining in with a rabbit isotype control (FIG. 2). Histological staining in human tumor array with anti-TnC clone 18D4 corresponds to specific TnC stroma fibers. The TnC staining is expressed at higher levels in most tumor tissues compared to control normal pair tissue (FIG. 3). Histological staining in human tumor array with anti-TnC clone 11C7 corresponds to specific TnC stroma fibers. The TnC staining is expressed at higher levels in most tumor tissues compared to control normal pair tissue (FIG. 4).

Example 7

Preparation, Purification and Characterization of 4-1BB

DNA sequences encoding the ectodomains of human, mouse or cynomolgus 4-1BB (Table 27) were subcloned in frame with the human IgG1 heavy chain CH2 and CH3 domains on the knob (Merchant et al., 1998). An AcTEV protease cleavage site was introduced between an antigen ectodomain and the Fc of human IgG1. An Avi tag for directed biotinylation was introduced at the C-terminus of the antigen-Fc knob. Combination of the antigen-Fc knob chain containing the S354C/T366W mutations, with a Fc hole chain containing the Y349C/T366S/L368A/Y407V mutations allows generation of a heterodimer which includes a single copy of 4-1BB ectodomain containing chain, thus creating a monomeric form of Fc-linked antigen. Table 28 shows the cDNA and amino acid sequences of the antigen Fc-fusion constructs.

TABLE 27 Amino acid numbering of antigen ectodomains (ECD) and their origin SEQ ID NO: Construct Origin ECD SEQ ID NO: 85 human Synthetized aa 24-186 4-1BB ECD according to Q07011 SEQ ID NO: 86 cynomolgus isolated from aa 24-186 4-1BB ECD cynomolgus blood SEQ ID NO: 87 murine Synthetized aa 24-187 4-1BB ECD according to P20334

TABLE 28 cDNA and Amino acid sequences of monomeric antigen Fc(kih) fusion molecules SEQ ID NO: Antigen Sequence SEQ ID NO: 88 Nucleotide GACAAAACTCACACATGCCCACCGTGCCCAGCACC sequence TGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC Fc hole chain CCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGT GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG GTGTGCACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCA CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 89 Nucleotide CTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCAC sequence CTTCTGCGACAACAACCGGAACCAGATCTGCAGCC human 4-1BB CCTGCCCCCCCAACAGCTTCAGCTCTGCCGGCGGA antigen Fc knob CAGCGGACCTGCGACATCTGCAGACAGTGCAAGG chain GCGTGTTCAGAACCCGGAAAGAGTGCAGCAGCAC CAGCAACGCCGAGTGCGACTGCACCCCCGGCTTCC ATTGTCTGGGAGCCGGCTGCAGCATGTGCGAGCAG GACTGCAAGCAGGGCCAGGAACTGACCAAGAAGG GCTGCAAGGACTGCTGCTTCGGCACCTTCAACGAC CAGAAGCGGGGCATCTGCCGGCCCTGGACCAACT GTAGCCTGGACGGCAAGAGCGTGCTGGTCAACGG CACCAAAGAACGGGACGTCGTGTGCGGCCCCAGC CCTGCTGATCTGTCTCCTGGGGCCAGCAGCGTGAC CCCTCCTGCCCCTGCCAGAGAGCCTGGCCACTCTC CTCAGGTCGACGAACAGTTATATTTTCAGGGCGGC TCACCCAAATCTGCAGACAAAACTCACACATGCCC ACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC CTCATGATCTCCCGGACCCCTGAGGTCACATGCGT GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG CCCCGAGAACCACAGGTGTACACCCTGCCCCCATG CCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG TGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATCCGGAGGCCTGAACGACATCTTCGAGGCCCA GAAGATTGAATGGCACGAG SEQ ID NO: 90 Nucleotide TTGCAGGATCTGTGTAGTAACTGCCCAGCTGGTAC sequence ATTCTGTGATAATAACAGGAGTCAGATTTGCAGTC cynomolgus 4- CCTGTCCTCCAAATAGTTTCTCCAGCGCAGGTGGA 1BB antigen CAAAGGACCTGTGACATATGCAGGCAGTGTAAAG Fc knob chain GTGTTTTCAAGACCAGGAAGGAGTGTTCCTCCACC AGCAATGCAGAGTGTGACTGCATTTCAGGGTATCA CTGCCTGGGGGCAGAGTGCAGCATGTGTGAACAG GATTGTAAACAAGGTCAAGAATTGACAAAAAAAG GTTGTAAAGACTGTTGCTTTGGGACATTTAATGAC CAGAAACGTGGCATCTGTCGCCCCTGGACAAACTG TTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGA CGAAGGAGAGGGACGTGGTCTGCGGACCATCTCC AGCCGACCTCTCTCCAGGAGCATCCTCTGCGACCC CGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCG CAGGTCGACGAACAGTTATATTTTCAGGGCGGCTC ACCCAAATCTGCAGACAAAACTCACACATGCCCAC CGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCT CATGATCTCCCGGACCCCTGAGGTCACATGCGTGG TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC TGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC CCGAGAACCACAGGTGTACACCCTGCCCCCATGCC GGGATGAGCTGACCAAGAACCAGGTCAGCCTGTG GTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCG CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCG ACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT CATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAA ATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGA AGATTGAATGGCACGAG SEQ ID NO: 91 Nucleotide GTGCAGAACAGCTGCGACAACTGCCAGCCCGGCA sequence murine CCTTCTGCCGGAAGTACAACCCCGTGTGCAAGAGC 4-1BB antigen Fc TGCCCCCCCAGCACCTTCAGCAGCATCGGCGGCCA knob chain GCCCAACTGCAACATCTGCAGAGTGTGCGCCGGCT ACTTCCGGTTCAAGAAGTTCTGCAGCAGCACCCAC AACGCCGAGTGCGAGTGCATCGAGGGCTTCCACTG CCTGGGCCCCCAGTGCACCAGATGCGAGAAGGAC TGCAGACCCGGCCAGGAACTGACCAAGCAGGGCT GTAAGACCTGCAGCCTGGGCACCTTCAACGACCAG AACGGGACCGGCGTGTGCCGGCCTTGGACCAATTG CAGCCTGGACGGGAGAAGCGTGCTGAAAACCGGC ACCACCGAGAAGGACGTCGTGTGCGGCCCTCCCGT GGTGTCCTTCAGCCCTAGCACCACCATCAGCGTGA CCCCTGAAGGCGGCCCTGGCGGACACTCTCTGCAG GTCCTGGTCGACGAACAGTTATATTTTCAGGGCGG CTCACCCAAATCTGCAGACAAAACTCACACATGCC CACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC CCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGT CAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG CCCCGAGAACCACAGGTGTACACCCTGCCCCCATG CCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG TGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATCCGGAGGCCTGAACGACATCTTCGAGGCCCA GAAGATTGAATGGCACGAG SEQ ID NO: 92 Fc hole chain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLS CAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 93 human 4-1BB LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQR antigen Fc knob TCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGA chain GCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGI CRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPG ASSVTPPAPAREPGHSPQVDEQLYFQGGSPKSADKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKSGGLNDIFEAQKIEWHE SEQ ID NO: 94 cynomolgus 4- LQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQR 1BB antigen TCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGA Fc knob chain ECSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGI CRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPG ASSATPPAPAREPGHSPQVDEQLYFQGGSPKSADKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKSGGLNDIFEAQKIEWHE SEQ ID NO: 95 murine 4-1BB VQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPN antigen Fc knob CNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQ chain CTRCEKDCRPGQELTKQGCKTCSLGTFNDQNGTGVC RPWTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTT ISVTPEGGPGGHSLQVLVDEQLYFQGGSPKSADKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKSGGLNDIFEAQKIEWHE

All 4-1BB-Fc-fusion molecule encoding sequences were cloned into a plasmid vector, which drives expression of the insert from an MPSV promoter and contains a synthetic polyA signal sequence located at the 3′ end of the CDS. In addition, the vector contains an EBV OriP sequence for episomal maintenance of the plasmid.

For preparation of the biotinylated monomeric antigen/Fc fusion molecules, exponentially growing suspension HEK293 EBNA cells were co-transfected with three vectors encoding the two components of fusion protein (knob and hole chains) as well as BirA, an enzyme necessary for the biotinylation reaction. The corresponding vectors were used at a 2:1:0.05 ratio (“antigen ECD-AcTEV-Fc knob”:“Fc hole”:“BirA”).

For protein production in 500 ml shake flasks, 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 minutes at 210 g, and the supernatant was replaced by pre-warmed CD CHO medium. Expression vectors were resuspended in 20 mL of CD CHO medium containing 200 μg of vector DNA. After addition of 540 μL of polyethylenimine (PEI), the solution was vortexed for 15 seconds and incubated for 10 minutes at room temperature. Afterwards, cells were mixed with the DNA/PEI solution, transferred to a 500 mL shake flask and incubated for 3 hours at 37° C. in an incubator with a 5% CO₂ atmosphere. After the incubation, 160 mL of F17 medium was added and cells were cultured for 24 hours. The production medium was supplemented with 5 mM kifunensine. One day after transfection, 1 mM valproic acid and 7% Feed 1 with supplements were added to the culture. After 7 days of culturing, the cell supernatant was collected by spinning down cells for 15 min at 210 g. The solution was sterile filtered (0.22 μm filter), supplemented with sodium azide to a final concentration of 0.01% (w/v), and kept at 4° C.

Secreted proteins were purified from cell culture supernatants by affinity chromatography using Protein A, followed by size exclusion chromatography. For affinity chromatography, the supernatant was loaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40 mL 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride containing buffer (pH 7.5). The bound protein was eluted using a linear pH-gradient of sodium chloride (from 0 to 500 mM) created over 20 column volumes of 20 mM sodium citrate, 0.01% (v/v) Tween-20, pH 3.0. The column was then washed with 10 column volumes of 20 mM sodium citrate, 500 mM sodium chloride, 0.01% (v/v) Tween-20, pH 3.0.

The pH of collected fractions was adjusted by adding 1/40 (v/v) of 2M Tris, pH8.0. The protein was concentrated and filtered prior to loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 2 mM MOPS, 150 mM sodium chloride, 0.02% (w/v) sodium azide solution of pH 7.4.

For affinity determination to the human receptor, the ectodomain of human 4-1BB was also subcloned in frame with an avi (GLNDIFEAQKIEWHE (SEQ ID NO: 225)) and a hexahistidine tag.

Protein production was performed as described above for the Fc-fusion protein. Secreted proteins were purified from cell culture supernatants by chelating chromatography, followed by size exclusion chromatography. The first chromatographic step was performed on a NiNTA Superflow Cartridge (5 ml, Qiagen) equilibrated in 20 mM sodium phosphate, 500 nM sodium chloride, pH7.4. Elution was performed by applying a gradient over 12 column volume from 5% to 45% of elution buffer (20 mM sodium phosphate, 500 nM sodium chloride, 500 mM Imidazole, pH7.4). The protein was concentrated and filtered prior to loading on a HiLoad Superdex 75 column (GE Healthcare) equilibrated with 2 mM MOPS, 150 mM sodium chloride, 0.02% (w/v) sodium azide solution of pH 7.4 (Table 29).

TABLE 29 Sequences of monomeric human 4-1BB His molecule SEQ ID NO: antigen Sequence SEQ ID NO: 96 nucleotide CTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCA sequence CCTTCTGCGACAACAACCGGAACCAGATCTGCAG human CCCCTGCCCCCCCAACAGCTTCAGCTCTGCCGGC 4-1BB His GGACAGCGGACCTGCGACATCTGCAGACAGTGC AAGGGCGTGTTCAGAACCCGGAAAGAGTGCAGC AGCACCAGCAACGCCGAGTGCGACTGCACCCCC GGCTTCCATTGTCTGGGAGCCGGCTGCAGCATGT GCGAGCAGGACTGCAAGCAGGGCCAGGAACTGA CCAAGAAGGGCTGCAAGGACTGCTGCTTCGGCA CCTTCAACGACCAGAAGCGGGGCATCTGCCGGCC CTGGACCAACTGTAGCCTGGACGGCAAGAGCGT GCTGGTCAACGGCACCAAAGAACGGGACGTCGT GTGCGGCCCCAGCCCTGCTGATCTGTCTCCTGGG GCCAGCAGCGTGACCCCTCCTGCCCCTGCCAGAG AGCCTGGCCACTCTCCTCAGGTCGACGAACAGTT ATATTTTCAGGGCGGCTCAGGCCTGAACGACATC TTCGAGGCCCAGAAGATCGAGTGGCACGAGGCT CGAGCTCACCACCATCACCATCAC SEQ ID NO: 97 human LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQ 4-1BB His RTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCL GAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQK RGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPAD LSPGASSVTPPAPAREPGHSPQVDEQLYFQGGSGLN DIFEAQKIEWHEARAHHHHHH

Example 8 Preparation of TnC Targeted Split Trimeric 4-1BB Ligand Fc Fusion Molecules

The DNA sequence encoding part of the ectodomain (amino acid 71-254 and 71-248) of human 4-1BB ligand was synthesized according to the P41273 sequence of Uniprot database. The TnC binder used to target the trimeric 4-1BB ligand was clone 18D4.

Construct 6.1: Monovalent TnC (18D4) Targeted Split Trimeric 4-1BB Ligand (71-254) Fc (kih) Fusion with CH-CL Cross and Charged Residues

A polypeptide containing two ectodomains of 4-1BB ligand (71-254), separated by (G4S)2 (SEQ ID NO: 150) linkers, and fused to the human IgG1-CL domain, was cloned as depicted in FIG. 5A: human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human CL.

A polypeptide containing one ectodomain of 4-1BB ligand (71-254) and fused to the human IgG1-CH domain, was cloned as described in FIG. 5B: human 4-1BB ligand, (G4S)2 connector, human CH.

To improve correct pairing the following mutations have been introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused to human CL, E123R and Q124K. In the monomeric 4-1BB ligand fused to human CH1, K147E and K213E.

The variable region of heavy and light chain DNA sequences encoding a binder specific for Tenescin (TnC), clone 18D4, were subcloned in frame with either the constant heavy chain of the hole or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced in the constant region of the knob and hole heavy chains to abrogate binding to Fc gamma receptors (as described in International Patent Appl. Publ. No. WO 2012/130831).

Combination of the dimeric ligand-Fc knob chain containing the S354C/T366W mutations, the monomeric CH1 fusion, the targeted anti-TnC-Fc hole chain containing the Y349C/T366S/L368A/Y407V mutations and the anti-TnC light chain (as described e.g. in U.S. Pat. Nos. 5,731,168; 7,695,936) allows generation of a heterodimer, which includes an assembled trimeric 4-1BB ligand and a TnC binding Fab (FIG. 2).

Table 30 and Table 31 show, respectively, the cDNA and amino acid sequences of the monovalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion with CH-CL cross and charged residues (construct 6.1).

TABLE 30 Base pair sequences of monovalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion containing CH-CL cross with charged residues (construct 6.1). SEQ ID NO Construct Sequence SEQ ID NO: 98 Dimeric ligand AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGC (71-254)-CL* TGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTC Fc knob chain AGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGC CCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGG CGTGTCACTGACAGGCGGCCTGAGCTACAAAGAG GACACCAAAGAACTGGTGGTGGCCAAGGCCGGCG TGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGA GTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCT GGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTG GCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCT CCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGG GTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCC AGAGGCTGGGAGTGCATCTGCACACAGAGGCCAG GGCTAGACACGCCTGGCAGCTGACACAGGGCGCT ACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGAT TCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCG GAGGCGGATCTGGCGGCGGAGGATCTAGAGAGGG ACCCGAACTGTCCCCTGACGATCCAGCCGGGCTGC TGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTG GCTCAGAATGTGCTGCTGATTGACGGACCTCTGAG CTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCC TGACTGGGGGACTGTCCTACAAAGAAGATACAAA AGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATG TGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCT GGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCA TCTGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTG CACTGGCTCTGACTGTGGACCTGCCACCAGCCTCT AGCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAG GACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTG GGAGTGCATCTGCATACTGAAGCCAGAGCCCGGC ATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTG GGACTGTTTCGCGTGACACCTGAGATCCCTGCCGG ACTGCCAAGCCCTAGATCAGAAGGGGGCGGAGGT TCCGGAGGGGGAGGATCTCGTACGGTGGCTGCACC ATCTGTCTTTATCTTCCCACCCAGCGACCGGAAGC TGAAGTCTGGCACAGCCAGCGTCGTGTGCCTGCTG AATAACTTCTACCCCCGCGAGGCCAAGGTGCAGTG GAAGGTGGACAATGCCCTGCAGAGCGGCAACAGC CAGGAAAGCGTGACCGAGCAGGACAGCAAGGACT CCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCACAAGGTGTACGCCT GCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTG ACCAAGAGCTTCAACCGGGGCGAGTGCGACAAGA CCCACACCTGTCCTCCATGCCCTGCCCCTGAAGCT GCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAA GCCCAAGGACACCCTGATGATCAGCCGGACCCCTG AAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAG GACCCTGAAGTGAAGTTCAATTGGTACGTGGACGG CGTGGAAGTGCACAATGCCAAGACCAAGCCGCGG GAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAG CCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCATGCCGGGATGAGCTGACCAAGAAC CAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAGCCGGAGAACAACTACAAGACCACGCCTC CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA SEQ ID NO: 99 Monomeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGC ligand (71-254)- TGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTC CH1* AGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGC CCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGG CGTGTCACTGACAGGCGGCCTGAGCTACAAAGAG GACACCAAAGAACTGGTGGTGGCCAAGGCCGGCG TGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGA GTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCT GGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTG GCGCTGCAGCTCTGGCTCTGACAGTGGATCTGCCT CCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGG GTTTCAAGGCCGGCTGCTGCACCTGTCTGCCGGCC AGAGACTGGGAGTGCATCTGCACACAGAGGCCAG AGCCAGGCACGCCTGGCAGCTGACACAGGGCGCT ACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGAT TCCTGCCGGCCTGCCTAGCCCTAGATCTGAAGGCG GCGGAGGTTCCGGAGGCGGAGGATCTGCTAGCAC AAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCA GCAAGAGCACATCTGGCGGAACAGCCGCCCTGGG CTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGA CCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGC GTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGG CCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCA GCAGCTCTCTGGGCACCCAGACCTACATCTGCAAC GTGAACCACAAGCCCAGCAACACCAAGGTGGACG AGAAGGTGGAACCCAAGTCCTGC SEQ ID NO: 100 anti- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAA TnC(18D4) Fc GAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGG hole chain CCTCCGGAGGCACATTCAGCAGCTACGCTATAAGC TGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGT GGATGGGAGGGATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAAGTTCCAGGGCAGGGTCACCA TTACTGCAGACAAATCCACGAGCACAGCCTACATG GAGCTGAGCAGCCTGAGATCTGAGGACACCGCCG TGTATTACTGTGCGAAAGGTAACTTCTACGGTGGT CTGGACTACTGGGGCCAAGGGACCACCGTGACCGT CTCCTCAGCTAGCACCAAGGGCCCCTCCGTGTTCC CCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGG CACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACT TCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGA GCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGT GCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCG TGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCCAGCAA CACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGC TGCGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCT TCCCCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGT GAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC TCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCT GACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAG GTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGA TGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain *for charged residues

TABLE 31 Amino acid sequences of monovalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion containing CH-CL cross with charged residues (construct 6.1). SEQ ID NO Construct Sequence SEQ ID NO: 102 Dimeric ligand REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP (71-254)-CL* LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY Fc knob chain YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGM FAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSL ALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGF QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSRTVAAPS VFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTIS KAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK SEQ ID NO: 103 Monomeric REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP ligand (71-254)- LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY CH1* YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSEGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGT AALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD EKVEPKSC SEQ ID NO: 104 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW TnC(18D4) Fc VRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA hole chain DKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain *for charged residues

Construct 6.2: Monovalent TnC (18D4) Targeted Split Trimeric 4-1BB Ligand (71-254) Fc (kih) Fusion Containing CH-CL Cross without Charged Residues

A polypeptide containing two ectodomains of 4-1BB ligand (71-254), separated by (G4S)2 (SEQ ID NO: 150) linkers, and fused to the human IgG1-CL domain, was cloned as depicted in FIG. 7A: human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human CL.

A polypeptide containing one ectodomain of 4-1BB ligand (71-254) and fused to the human IgG1-CH domain, was cloned as described in FIG. 7B: human 4-1BB ligand, (G4S)2 connector, human CH.

The variable region of heavy and light chain DNA sequences encoding a binder specific for TnC, clone 18D4, were subcloned in frame with either the constant heavy chain of the hole or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced in the constant region of the knob and hole heavy chains to abrogate binding to Fc gamma receptors (patent accession number).

Combination of the dimeric ligand-Fc knob chain containing the S354C/T366W mutations, the monomeric CH1 fusion, the targeted anti-TnC-Fc hole chain containing the Y349C/T366S/L368A/Y407V mutations and the anti-TnC light chain allows generation of a heterodimer, which includes an assembled trimeric 4-1BB ligand and a TnC binding Fab (FIG. 8).

Table 32 and Table 33 show, respectively, the cDNA and amino acid sequences of the monovalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion containing CH-CL cross without charged residues (construct 6.2).

TABLE 32 Base pair sequences of monovalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion containing CH-CL cross without charged residues (construct 6.2). SEQ ID NO Construct Sequence SEQ ID NO: 106 Dimeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71-254)- GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGC CL Fc TGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCT knob chain GTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCA CTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAA GAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGT TCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGA AGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAG CCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCAC TGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCG GAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCAC CTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACA CAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACAC AGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCC CGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAA GGCGGAGGCGGATCTGGCGGCGGAGGATCTAGAGAG GGACCCGAACTGTCCCCTGACGATCCAGCCGGGCTGC TGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGC TCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGG TACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTG GGGGACTGTCCTACAAAGAAGATACAAAAGAACTGG TGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCA GCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTC AGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTG CGCTCTGCTGCTGGCGCAGCTGCACTGGCTCTGACTG TGGACCTGCCACCAGCCTCTAGCGAGGCCAGAAACA GCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAG CGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGA AGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGG GGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAG ATCCCTGCCGGACTGCCAAGCCCTAGATCAGAAGGG GGCGGAGGTTCCGGAGGGGGAGGATCTCGTACGGTG GCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGA ACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTG CTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGT GGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCC AGGAATCTGTGACAGAACAGGACTCCAAGGACAGCA CCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCT GATTATGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTGACAAGACCCACACCTGTC CCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCT GTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGA TGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGT GGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAAT TGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAG ACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCA ACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA CACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAA CCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTAT CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAA SEQ ID NO: 107 Monomeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71- GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGC 254)-CH1 TGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCT GTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCA CTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAA GAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGT TCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGA AGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAG CCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCTC TGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCG GAATAGCGCATTTGGGTTTCAAGGCCGGCTGCTGCAC CTGTCTGCCGGCCAGAGACTGGGAGTGCATCTGCACA CAGAGGCCAGAGCCAGGCACGCCTGGCAGCTGACAC AGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCC CGAGATTCCTGCCGGCCTGCCTAGCCCTAGATCTGAA GGCGGCGGAGGTTCCGGAGGCGGAGGATCTGCTAGC ACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTC CAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGC CTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGT CCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATAC TTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGC CTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTG GGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCC AAGTCTTGT SEQ ID NO: 98 anti- See Table 30 TnC(18D4) Fc hole chain SEQ ID NO: 99 anti- See Table 30 TnC(18D4) light chain

TABLE 33 Amino acid sequences of monovalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion containing CH-CL cross without charged residues (construct 6.2). SEQ ID NO Construct Sequence SEQ ID NO: 108 Dimeric ligand REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS (71-254)-CL WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF Fc knob chain QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTV DLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGS GGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAA LALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHL HTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEG GGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPI EKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 109 Monomeric REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS ligand (71-254)- WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF CH1 QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTV DLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGS GGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 104 anti- See Table 31 TnC(18D4) Fc hole chain SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Construct 6.3: Bivalent TnC (18D4) Targeted Split Trimeric 4-1BB Ligand (71-254) Fc (kih) Fusion

A polypeptide containing two ectodomains of 4-1BB ligand (71-254), separated by (G4S)2 (SEQ ID NO: 150) linkers was fused to the C-terminus of human IgG1 Fc hole chain, as depicted in FIG. 9A: human IgG1 Fc hole, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand.

A polypeptide containing one ectodomain of 4-1BB ligand (71-254) and fused to the C-terminus of human IgG1 Fc knob chain as described in FIG. 9B: human IgG1 Fc knob, (G4S)2 connector, human 4-1BB ligand.

The variable region of heavy and light chain DNA sequences encoding a binder specific for TnC, clone 18D4, were subcloned in frame with either the constant heavy chain of the hole, the knob or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced in the constant region of the knob and hole heavy chains to abrogate binding to Fc gamma receptors (described in International Patent Appl. Publ. No. WO 2012/130831).

Combination of the anti-TnC huIgG1 hole dimeric ligand chain containing the Y349C/T366S/L368A/Y407V mutations, the anti-TnC huIgG1 knob monomeric ligand chain containing the S354C/T366W mutations and the anti-TnC light chain allows generation of a heterodimer, which includes an assembled trimeric 4-1BB ligand and two TnC binding Fabs (FIG. 10).

Table 34 and Table 35 show, respectively, the cDNA and amino acid sequences of the bivalent TnC (18D4)-targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion (construct 6.3).

TABLE 34 Base pair sequences of bivalent TnC(18D4) targeted split trimeric 4-1BB ligand Fc (kih) PGLALA fusion (construct 6.3) SEQ IO NO Construct Sequence SEQ ID NO: 110 anti-TnC(18D4) CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGA Fc hole dimeric AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG ligand (71-254) GCCTCCGGAGGCACATTCAGCAGCTACGCTATAAG chain CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGAGGGATCATCCCTATCTTTGGTACAGC AAACTACGCACAGAAGTTCCAGGGCAGGGTCACC ATTACTGCAGACAAATCCACGAGCACAGCCTACAT GGAGCTGAGCAGCCTGAGATCTGAGGACACCGCC GTGTATTACTGTGCGAAAGGTAACTTCTACGGTGG TCTGGACTACTGGGGCCAAGGGACCACCGTGACC GTCTCCTCAGCTAGCACCAAGGGCCCCTCCGTGTT CCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGC GGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTA CTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCG GAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCC GTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAG CGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCC AGACCTACATCTGCAACGTGAACCACAAGCCCAG CAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG AGCTGCGACAAAACTCACACATGCCCACCGTGCCC AGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATC TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTGCACCCTGCCCCCATCCCGGGATG AGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGC AGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGC GGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTG AGCTGAGCCCCGATGATCCTGCTGGACTGCTGGAC CTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCA GAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGT ACAGCGATCCTGGACTGGCTGGCGTGTCACTGACA GGCGGCCTGAGCTACAAAGAGGACACCAAAGAAC TGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTC TTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCG AAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTG CAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCT GGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCG AGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAG GCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAG TGCATCTGCACACAGAGGCCAGGGCTAGACACGC CTGGCAGCTGACACAGGGCGCTACAGTGCTGGGC CTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCT GCCTTCTCCAAGAAGCGAAGGCGGAGGCGGATCT GGCGGCGGAGGATCTAGAGAGGGACCCGAACTGT CCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGA CAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGT GCTGCTGATTGACGGACCTCTGAGCTGGTACTCCG ACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGG ACTGTCCTACAAAGAAGATACAAAAGAACTGGTG GTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCA GCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGC TCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCC ACTGCGCTCTGCTGCTGGCGCAGCTGCACTGGCTC TGACTGTGGACCTGCCACCAGCCTCTAGCGAGGCC AGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCT GCATCTGAGCGCCGGACAGCGCCTGGGAGTGCAT CTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCA GCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTC GCGTGACACCTGAGATCCCTGCCGGACTGCCAAGC CCTAGATCAGAA SEQ ID NO: 111 anti-TnC(18D4) CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGA Fc knob AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG monomeric GCCTCCGGAGGCACATTCAGCAGCTACGCTATAAG ligand (71-254) CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG chain TGGATGGGAGGGATCATCCCTATCTTTGGTACAGC AAACTACGCACAGAAGTTCCAGGGCAGGGTCACC ATTACTGCAGACAAATCCACGAGCACAGCCTACAT GGAGCTGAGCAGCCTGAGATCTGAGGACACCGCC GTGTATTACTGTGCGAAAGGTAACTTCTACGGTGG TCTGGACTACTGGGGCCAAGGGACCACCGTGACC GTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTT CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGG GCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCC AGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAAAGTTGAGCCCAAA TCTTGTGACAAAACTCACACATGCCCACCGTGCCC AGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATC TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGCCCCCCTGCAGAGATG AGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTG GTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGA GTGGGAGAGCAACGGCCAGCCTGAGAACAACTAC AAGACCACCCCCCCTGTGCTGGACAGCGACGGCA GCTTCTTCCTGTACTCCAAACTGACCGTGGACAAG AGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCA GCGTGATGCACGAGGCCCTGCACAACCACTACAC CCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGC GGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGC CCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCT GGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGG CCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCC TGGTACAGCGATCCTGGACTGGCTGGCGTGTCACT GACAGGCGGCCTGAGCTACAAAGAGGACACCAAA GAACTGGTGGTGGCCAAGGCCGGCGTGTACTACG TGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCC GGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCA TCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAG CTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGC TCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGG CAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGG GAGTGCATCTGCACACAGAGGCCAGGGCTAGACA CGCCTGGCAGCTGACACAGGGCGCTACAGTGCTG GGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGG CCTGCCTTCTCCAAGAAGCGAA SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

TABLE 35 Amino acid sequences of bivalent TnC(18D4) targeted split trimeric 4-1BB ligand Fc (kih) PGLALA fusion (construct 6.3) SEQ ID NO Construct Sequence SEQ ID NO: 112 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS TnC(18D4) Fc WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI hole dimeric TADKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGL ligand (71-254) DYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA chain ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSR EGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAG AAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQ RLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIP AGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDL RQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGE GSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEA RNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE SEQ ID NO: 113 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS TnC(18D4) Fc WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI knob TADKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGL monomeric DYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ligand (71-254) ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ chain SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTL PPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGS REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDG PLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAG AAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQ RLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIP AGLPSPRSE SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Construct 6.4: Monovalent TnC (18D4) Targeted Split Trimeric 4-1BB Ligand (71-248) Fc (kih) Fusion Containing CH-CL Cross with Charged Residues

A polypeptide containing two ectodomains of 4-1BB ligand (71-248), separated by (G4S)2 (SEQ ID NO: 150) linkers, and fused to the human IgG1-CL domain, was cloned as depicted in FIG. 11A: human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human CL.

A polypeptide containing one ectodomain of 4-1BB ligand (71-248) and fused to the human IgG1-CH domain, was cloned as described in FIG. 11B: human 4-1BB ligand, (G4S)2 connector, human CH.

To improve correct pairing the following mutations have been introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused to human CL, E123R and Q124K. In the monomeric 4-1BB ligand fused to human CH1, K147E and K213E.

The variable region of heavy and light chain DNA sequences encoding a binder specific for TnC, clone 18D4, were subcloned in frame with either the constant heavy chain of the hole, the knob or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced in the constant region of the knob and hole heavy chains to abrogate binding to Fc gamma receptors (patent accession number).

Combination of the dimeric ligand-Fc knob chain containing the S354C/T366W mutations, the monomeric CH1 fusion, the targeted anti-TnC-Fc hole chain containing the Y349C/T366S/L368A/Y407V mutations and the anti-TnC light chain allows generation of a heterodimer, which includes an assembled trimeric 4-1BB ligand and a TnC binding Fab (FIG. 12).

Table 36 and Table 37 show, respectively, the cDNA and amino acid sequences of the monovalent TnC targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross with charged residues (construct 6.4).

TABLE 36 Base pair sequences of monovalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross with charged residues (construct 6.4). SEQ ID NO Construct Sequence SEQ ID NO: 114 Dimeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71- GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGC 248)-CL* Fc TGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCT knob chain GTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCA CTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAA GAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGT TCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGA AGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAG CCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCAC TGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCG GAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCAC CTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACA CAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACAC AGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCC CGAGATTCCAGCCGGACTGGGAGGCGGCGGATCTGG CGGCGGAGGATCTAGAGAAGGACCCGAGCTGTCCCC TGACGATCCAGCCGGGCTGCTGGATCTGAGACAGGG AATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTG ATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGC TGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAA AGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGG GGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGG GTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGG CTCTGCATCTGCAGCCACTGCGCTCTGCAGCAGGGGC TGCAGCACTGGCCCTGACTGTGGACCTGCCCCCAGCT TCTTCCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAG GACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGG GAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATG CTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACT GTTTCGCGTGACACCTGAGATCCCCGCTGGACTGGGC GGAGGCGGTTCCGGAGGGGGAGGATCTCGTACGGTG GCTGCACCATCTGTCTTTATCTTCCCACCCAGCGACC GGAAGCTGAAGTCTGGCACAGCCAGCGTCGTGTGCCT GCTGAATAACTTCTACCCCCGCGAGGCCAAGGTGCAG TGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGC CAGGAAAGCGTGACCGAGCAGGACAGCAAGGACTCC ACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAG GCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAA GTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGA GCTTCAACCGGGGCGAGTGCGACAAGACCCACACCT GTCCTCCATGCCCTGCCCCTGAAGCTGCTGGCGGCCC TAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACC CTGATGATCAGCCGGACCCCTGAAGTGACCTGCGTGG TGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAATGC CAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC TCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG GTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCA AGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTT CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA SEQ ID NO: 115 Monomeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71- GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGC 248)-CH1* TGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCT GTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCA CTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAA GAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGT TCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGA AGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAG CCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCAC TGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCG GAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCAC CTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACA CAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACAC AGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCC CGAGATTCCAGCCGGACTGGGAGGCGGAGGTTCCGG AGGCGGAGGATCTGCTAGCACAAAGGGCCCCAGCGT GTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGC GGAACAGCCGCCCTGGGCTGCCTGGTGGAAGATTACT TCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGC CCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTG CAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGA CAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACAT CTGCAACGTGAACCACAAGCCCAGCAACACCAAGGT GGACGAGAAGGTGGAACCCAAGTCCTGC SEQ ID NO: 100 anti- See Table 30 TnC(18D4) Fc hole chain SEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain *charged residues

TABLE 37 Amino acid sequences of monovalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross with charged residues (construct 6.4). SEQ ID NO Construct Sequence SEQ ID NO: 116 Dimeric REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS ligand (71- WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF 248)-CL* Fc QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTV knob chain DLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSR EGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSW YSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQ LELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVD LPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARA RHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSRT VAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQP REPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 117 Monomeric REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS ligand (71- WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF 248)-CH1* QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTV DLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGS ASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDEKVEPKSC SEQ ID NO: 104 anti- See Table 31 TnC(18D4) Fc hole chain SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain *charged residues

Construct 6.5: Monovalent TnC (18D4) Targeted Split Trimeric 4-1BB Ligand (71-248) Fc (kih) Fusion Containing CH-CL Cross without Charged Residues

A polypeptide containing two ectodomains of 4-1BB ligand (71-248), separated by (G4S)2 (SEQ ID NO: 150) linkers, and fused to the human IgG1-CL domain, was cloned as depicted in FIG. 13A: human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human CL.

A polypeptide containing one ectodomain of 4-1BB ligand (71-248) and fused to the human IgG1-CH domain, was cloned as described in FIG. 13B: human 4-1BB ligand, (G4S)2 connector, human CH.

The variable region of heavy and light chain DNA sequences encoding a binder specific for TnC, clone 18D4, were subcloned in frame with either the constant heavy chain of the hole, the knob or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced in the constant region of the knob and hole heavy chains to abrogate binding to Fc gamma receptors (patent accession number).

Combination of the dimeric ligand-Fc knob chain containing the S354C/T366W mutations, the monomeric CH1 fusion, the targeted anti-TnC-Fc hole chain containing the Y349C/T366S/L368A/Y407V mutations and the anti-TnC light chain allows generation of a heterodimer, which includes an assembled trimeric 4-1BB ligand and a TnC binding Fab (FIG. 14).

Table 38 and Table 39 show, respectively, the cDNA and amino acid sequences of the monovalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-248) with charged residues in the CH-CL cross Fc (kih) fusion (construct 6.5).

TABLE 38 Base pair sequences of monovalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross without charged residues (construct 6.5). SEQ ID NO Construct Sequence SEQ ID NO: 118 Dimeric ligand AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT (71-248)-CL GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAG Fc knob chain CTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCC CTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTG TCACTGACAGGCGGCCTGAGCTACAAAGAGGACAC CAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACT ACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGG CCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGC ATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTG CTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCA GCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCA GGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAG TGCATCTGCACACAGAGGCCAGGGCTAGACACGCCT GGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGT TCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGA GGCGGCGGATCTGGCGGCGGAGGATCTAGAGAAGG ACCCGAGCTGTCCCCTGACGATCCAGCCGGGCTGCT GGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGC TCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTG GTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGAC TGGGGGACTGTCCTACAAAGAAGATACAAAAGAAC TGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTT TTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAG GGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAG CCACTGCGCTCTGCAGCAGGGGCTGCAGCACTGGCC CTGACTGTGGACCTGCCCCCAGCTTCTTCCGAGGCC AGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTG CATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTG CATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTG ACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTG ACACCTGAGATCCCCGCTGGACTGGGCGGAGGCGG TTCCGGAGGGGGAGGATCTCGTACGGTGGCCGCTCC CTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTG AAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAAC AATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAA GTGGATAACGCACTGCAGTCCGGCAACTCCCAGGA ATCTGTGACAGAACAGGACTCCAAGGACAGCACCT ACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTG ATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCA CCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTGACAAGACCCACACCTGT CCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTT CTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCC TGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGG TGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGT TCAATTGGTACGTGGACGGCGTGGAAGTGCACAATG CCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATGCCGGGATGAGC TGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTAAA SEQ ID NO: 119 Monomeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71-248)- GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAG CH1 CTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCC CTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTG TCACTGACAGGCGGCCTGAGCTACAAAGAGGACAC CAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACT ACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGG CCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGC ATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTG CTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCA GCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCA GGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAG TGCATCTGCACACAGAGGCCAGGGCTAGACACGCCT GGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGT TCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGA GGCGGAGGTTCCGGAGGCGGAGGATCTGCTAGCAC CAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCC AAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGC CTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTG TCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCAT ACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACA GCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGC CTGGGCACCCAGACCTACATCTGCAACGTGAACCAC AAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGA ACCCAAGTCTTGT SEQ ID NO: 100 anti- See Table 30 TnC(18D4) Fc hole chain SEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain

TABLE 39 Amino acid sequences of monovalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross without charged residues (construct 6.5). SEQ ID NO Construct Sequence SEQ ID NO: 120 Dimeric ligand REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL (71-248)-CL SWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV Fc knob chain FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLH TEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSG GGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAG VYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGG GGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 121 Monomeric REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL ligand (71-248)- SWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV CH1 FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLH TEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSG GGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 104 anti- See Table 31 TnC(18D4) Fc hole chain SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Construct 6.6: Bivalent TnC (18D4) Targeted Split Trimeric 4-1BB Ligand (71-248) Fc (kih) Fusion

A polypeptide containing two ectodomains of 4-1BB ligand (71-248), separated by (G4S)2 (SEQ ID NO: 150) linkers was fused to the C-terminus of human IgG1 Fc hole chain, as depicted in FIG. 15A: human IgG1 Fc hole, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand.

A polypeptide containing one ectodomain of 4-1BB ligand (71-248) and fused to the C-terminus of human IgG1 Fc knob chain as described in FIG. 15B: human IgG1 Fc knob, (G4S)2 connector, human 4-1BB ligand.

The variable region of heavy and light chain DNA sequences encoding a binder specific for TnC, clone 18D4, were subcloned in frame with either the constant heavy chain of the hole, the knob or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced in the constant region of the knob and hole heavy chains to abrogate binding to Fc gamma receptors (patent accession number).

Combination of the anti-TnC huIgG1 hole dimeric ligand chain containing the Y349C/T366S/L368A/Y407V mutations, the anti-TnC huIgG1 knob monomeric ligand chain containing the S354C/T366W mutations and the anti-TnC light chain allows generation of a heterodimer, which includes an assembled trimeric 4-1BB ligand and two TnC binding Fabs (FIG. 16).

Table 40 and Table 41 show, respectively, the cDNA and amino acid sequences of the bivalent TnC (18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.6).

TABLE 40 Base pair sequences of bivalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.6) SEQ ID NO Construct Sequence SEQ ID NO: 122 anti- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAG TnC(18D4) Fc AAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCT hole dimeric CCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGG ligand (71-248) TGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGG chain GAGGGATCATCCCTATCTTTGGTACAGCAAACTACGC ACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGA CAAATCCACGAGCACAGCCTACATGGAGCTGAGCAG CCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCG AAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCC AAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCA AGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAA GAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCC TGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACC TTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCC TGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGG CACCCAGACCTACATCTGCAACGTGAACCACAAGCC CAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAA GAGCTGCGACAAAACTCACACATGCCCACCGTGCCC AGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGC CAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCT GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT CAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGG CGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCC CTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGA CCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCA GAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTAC AGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGC GGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTG GTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGC TGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTG GCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAG ATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTG GATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGC GCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTG CCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGG CCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCG CTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGAT TCCAGCAGGCCTGGGAGGCGGCGGATCTGGCGGCGG AGGATCTAGAGAAGGACCCGAGCTGTCCCCCGACGA TCCCGCTGGGCTGCTGGATCTGAGACAGGGCATGTTC GCTCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACG GACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAG GGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAG ATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGT ACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGT GGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTG CATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCA GCACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTT CCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGAC GCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAG TGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTG GCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTT CGCGTGACACCTGAGATCCCAGCCGGGCTC SEQ ID NO: 123 anti- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAG TnC(18D4) Fc AAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCT knob CCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGG monomeric (71- TGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGG 248) ligand GAGGGATCATCCCTATCTTTGGTACAGCAAACTACGC ACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGA CAAATCCACGAGCACAGCCTACATGGAGCTGAGCAG CCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCG AAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCC AAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCA AGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG GCACCCAGACCTACATCTGCAACGTGAATCACAAGC CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCA AATCTTGTGACAAAACTCACACATGCCCACCGTGCCC AGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGA GCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGC CAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGT GTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGC GATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCT GAGAACAACTACAAGACCACCCCCCCTGTGCTGGAC AGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCG TGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACT ACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAG GCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGC CCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGG ACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCC AGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTA CAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGT GGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAG CTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCT GGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGA GATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGT GGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAG CGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCT GCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAG GCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGC GCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAG ATTCCTGCCGGGCTC SEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain

TABLE 41 Amino acid sequences of bivalent TnC(18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.6) SEQ ID NO Construct Sequence SEQ ID NO: 124 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR TnC(18D4) Fc QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTS hole dimeric TAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTV ligand (71-248) TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE chain PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQP REPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSRE GPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLE LRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARH AWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGP ELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSD PGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPA SSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHA WQLTQGATVLGLFRVTPEIPAGL SEQ ID NO: 125 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR TnC(18D4) Fc QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTS knob TAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTV monomeric (71- TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE 248) ligand PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQP REPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGS REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTV DLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGL SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Constructs 6.11 and 6.12: Monovalent TnC (Knob) Targeted Trimeric C-Terminal 4-1BB Ligand Fc (kih) Fusion

A polypeptide containing two ectodomains of 4-1BB ligand, separated by (G4S)2 (SEQ ID NO: 150) linkers was subcloned in frame to the C-terminus of human IgG1 Fc hole or knob chain (Merchant, Zhu et al. 1998), as depicted in FIG. 17A: human IgG1 Fc, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand. A polypeptide containing one ectodomain of 4-1BB ligand was subcloned in frame to the C-terminus of human IgG1 Fc knob or hole chain as described in FIG. 17B: human IgG1 Fc, (G4S)2 connector, human 4-1BB ligand.

The variable region of heavy and light chain DNA sequences encoding a binder specific for TnC, clone 18D4, were subcloned in frame with either the constant heavy chain of the knob or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced in the constant region of the knob and hole heavy chains to abrogate binding to Fc gamma receptors (described in International Patent Appl. Publ. No. WO 2012/130831).

Combination of the huIgG1 Fc hole chain containing the Y349C/T366S/L368A/Y407V mutations, the anti-TnC huIgG1 knob chain containing the S354C/T366W mutations and the anti-TnC light chain allows generation of a heterodimer, which includes an assembled trimeric 4-1BB ligand and one TnC binding Fab (FIGS. 18A and 18B).

Table 42 and Table 43 show respectively, the cDNA and amino acid sequences of the monovalent targeted TnC (18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.11).

TABLE 42 Base pair sequences of bivalent TnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.11) SEQ ID NO Construct Sequence SEQ ID NO: 126 Fc hole dimeric GACAAAACTCACACATGCCCACCGTGCCCAGCACCT ligand (71-248) GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC chain CCAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCT GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG TCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAG AGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGAC TGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGG TGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGT CCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCAC TGACAGGCGGCCTGAGCTACAAAGAGGACACCAAA GAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTG TTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGC GAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTG CAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTG GCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAG GCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTG CTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCAT CTGCACACAGAGGCCAGGGCTAGACACGCCTGGCA GCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAG AGTGACCCCCGAGATTCCAGCAGGCCTGGGAGGCG GCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCC GAGCTGTCCCCCGACGATCCCGCTGGGCTGCTGGAT CTGAGACAGGGCATGTTCGCTCAGCTGGTGGCTCAG AATGTGCTGCTGATTGACGGACCTCTGAGCTGGTAC TCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGG GGACTGTCCTACAAAGAAGATACAAAAGAACTGGT GGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCA GCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCT CAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCAC TGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGA CTGTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGAA ACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATC TGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATA CTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTC AGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACAC CTGAGATCCCAGCCGGGCTC SEQ ID NO: 123 anti- See Table 40 TnC(18D4) Fc knob monomeric ligand (71-248) chain SEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain

TABLE 43 Amino acid sequences of bivalent TnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.11) SEQ ID NO Construct Sequence SEQ ID NO: 127 Fc hole dimeric DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV ligand (71-248) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ chain YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP IEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK ELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHL SAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTP EIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGM FAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKE DTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLAL HLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF RVTPEIPAGL SEQ ID NO: 125 anti- See Table 41 TnC(18D4) Fc knob monomeric ligand (71-248) chain SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Table 44 and Table 45 show, respectively, the cDNA and amino acid sequences of the monovalent targeted TnC (18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.12).

TABLE 44 Base pair sequences of monovalent targeted TnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.12) SEQ ID NO Construct Sequence SEQ ID NO: 128 Fc hole GACAAAACTCACACATGCCCACCGTGCCCAGCACCT monomeric GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC ligand (71-248) CCAAAACCCAAGGACACCCTCATGATCTCCCGGACC chain CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCT GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG TCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGC CGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCA GCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCC CCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGT GTCACTGACAGGCGGCCTGAGCTACAAAGAGGACA CCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTAC TACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTG GCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTG CATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCT GCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGC AGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGC AGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGA GTGCATCTGCACACAGAGGCCAGGGCTAGACACGC CTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCT GTTCAGAGTGACCCCCGAGATTCCTGCCGGGCTC SEQ ID NO: 129 anti- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAA TnC(18D4) Fc GAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGC knob dimeric CTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTG ligand (71-248) GGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGA chain TGGGAGGGATCATCCCTATCTTTGGTACAGCAAACT ACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTG CAGACAAATCCACGAGCACAGCCTACATGGAGCTG AGCAGCCTGAGATCTGAGGACACCGCCGTGTATTAC TGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTAC TGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCT AGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCC TCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTG GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG AATCACAAGCCCAGCAACACCAAGGTGGACAAGAA AGTTGAGCCCAAATCTTGTGACAAAACTCACACATG CCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGAC CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT GCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA GAACCACAGGTGTACACCCTGCCCCCCTGCAGAGAT GAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTG GTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAG TGGGAGAGCAACGGCCAGCCTGAGAACAACTACAA GACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCG GTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGAT GCACGAGGCCCTGCACAACCACTACACCCAGAAGT CCCTGAGCCTGAGCCCCGGCAGAGAGGGCCCTGAG CTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTG CGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAAC GTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGC GATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGC CTGAGCTACAAAGAGGACACCAAAGAACTGGTGGT GGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCT GGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTG GCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAG ATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGT GGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAG CGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTC TGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAG AGGCCAGGGCTAGACACGCCTGGCAGCTGACACAG GGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCC GAGATTCCAGCAGGCCTGGGAGGCGGCGGATCTGG CGGCGGAGGATCTAGAGAAGGACCCGAGCTGTCCC CCGACGATCCCGCTGGGCTGCTGGATCTGAGACAGG GCATGTTCGCTCAGCTGGTGGCTCAGAATGTGCTGC TGATTGACGGACCTCTGAGCTGGTACTCCGACCCAG GGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCT ACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAA GCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTG AGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGT GTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGC AGCAGGGGCTGCAGCACTGGCCCTGACTGTGGACCT GCCCCCAGCTTCTTCCGAGGCCAGAAACAGCGCCTT CGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGG ACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAG AGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAA CTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCC CAGCCGGGCTC SEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain

TABLE 45 Amino acid sequences of monovalent targeted TnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.12) SEQ ID NO Construct Sequence SEQ ID NO: 130 Fc hole DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV monomeric TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ ligand (71-248) YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP chain IEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLID GPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL SEQ ID NO: 131 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWV TnC(18D4) Fc RQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKS knob dimeric TSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQG ligand (71-248) TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD chain YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYY VFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAAL ALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHL HTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGS GGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLL IDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAG VYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Constructs 6.13 and 6.14: Monovalent TnC (Hole) Targeted Trimeric C-Terminal 4-1BB Ligand Fc (kih) Fusion

A polypeptide containing two ectodomains of 4-1BB ligand, separated by (G4S)2 (SEQ ID NO: 150) linkers was subcloned in frame to the C-terminus of human IgG1 Fc hole or knob chain (Merchant, Zhu et al. 1998), as depicted in FIG. 17A: human IgG1 Fc, (G4S)2 connector, human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand. A polypeptide containing one ectodomain of 4-1BB ligand was subcloned in frame to the C-terminus of human IgG1 Fc knob or hole chain as described in FIG. 17B: human IgG1 Fc, (G4S)2 connector, human 4-1BB ligand.

The variable region of heavy and light chain DNA sequences encoding a binder specific for TnC, clone 18D4, were subcloned in frame with either the constant heavy chain of the hole or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced in the constant region of the knob and hole heavy chains to abrogate binding to Fc gamma receptors (described in International Patent Appl. Publ. No. WO 2012/130831).

Combination of the anti-TnC huIgG1 Fc hole chain containing the Y349C/T366S/L368A/Y407V mutations, the huIgG1 knob chain containing the S354C/T366W mutations and the anti-TnC light chain allows generation of a heterodimer, which includes an assembled trimeric 4-1BB ligand and one TnC binding Fab (FIGS. 19A and 19B).

Table 46 and Table 47 show, respectively, the cDNA and amino acid sequences of the monovalent targeted TnC (18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.13).

TABLE 46 Base pair sequences of monovalent targeted TnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.13). SEQ ID NO Construct Sequence SEQ ID NO: 122 anti- See Table 40 TnC(18D4) Fc hole dimeric ligand (71-248) chain SEQ ID NO: 132 Fc knob GACAAAACTCACACATGCCCACCGTGCCCAGCACCT monomeric GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC ligand (71-248) CCAAAACCCAAGGACACCCTCATGATCTCCCGGACC chain CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGG TGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCA GCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAG CCTGAGAACAACTACAAGACCACCCCCCCTGTGCTG GACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTG ACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGT GTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAA CCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGG CGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGA GAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGA CTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTG GTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTG TCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCA CTGACAGGCGGCCTGAGCTACAAAGAGGACACCAA AGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGT GTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGG CGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCT GCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCT GGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGA GGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCT GCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCA TCTGCACACAGAGGCCAGGGCTAGACACGCCTGGC AGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCA GAGTGACCCCCGAGATTCCTGCCGGGCTC SEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain

TABLE 47 Amino acid sequences of monovalent targeted TnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.13). SEQ ID NO Construct Sequence SEQ ID NO: 124 anti- See Table 41 TnC(18D4) Fc hole dimeric ligand (71-248) chain SEQ ID NO: 133 Fc knob DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV monomeric TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ ligand (71-248) YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP chain IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK ELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHL SAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTP EIPAGL SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Table 48 and Table 49 show, respectively, the cDNA and amino acid sequences of the monovalent targeted TnC (18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.14).

TABLE 48 Base pair sequences of monovalent targeted TnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.14). SEQ ID NO Construct Sequence SEQ ID NO: 134 anti- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAA TnC(18D4) Fc GAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGC hole CTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTG monomeric GGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGA ligand (71-248) TGGGAGGGATCATCCCTATCTTTGGTACAGCAAACT chain ACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTG CAGACAAATCCACGAGCACAGCCTACATGGAGCTG AGCAGCCTGAGATCTGAGGACACCGCCGTGTATTAC TGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTAC TGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCT AGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCC AGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCT GGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGT GACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGG CGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGG CCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTC TAGCAGCCTGGGCACCCAGACCTACATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGACAAGA AGGTGGAGCCCAAGAGCTGCGACAAAACTCACACA TGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGG ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCAT CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC GAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGG ATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCG CAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAG CGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGA GCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGC AGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGC TGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATC CTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGA GCTACAAAGAGGACACCAAAGAACTGGTGGTGGCC AAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAA CTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCT GCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGAT CTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCA TTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCC GGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGC CAGGGCTAGACACGCCTGGCAGCTGACACAGGGCG CTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGA TTCCTGCCGGGCTC SEQ ID NO: 135 Fc knob GACAAAACTCACACATGCCCACCGTGCCCAGCACCT dimeric ligand GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC (71-248) chain CCAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGG TGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCA GCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAG CCTGAGAACAACTACAAGACCACCCCCCCTGTGCTG GACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTG ACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGT GTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAA CCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGG CGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGA GAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGA CTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTG GTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTG TCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCA CTGACAGGCGGCCTGAGCTACAAAGAGGACACCAA AGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGT GTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGG CGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCT GCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCT GGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGA GGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCT GCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCA TCTGCACACAGAGGCCAGGGCTAGACACGCCTGGC AGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCA GAGTGACCCCCGAGATTCCAGCAGGCCTGGGAGGC GGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACC CGAGCTGTCCCCCGACGATCCCGCTGGGCTGCTGGA TCTGAGACAGGGCATGTTCGCTCAGCTGGTGGCTCA GAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTA CTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGG GGGACTGTCCTACAAAGAAGATACAAAAGAACTGG TGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTC AGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGC TCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCA CTGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTG ACTGTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGA AACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCAT CTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCAT ACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACT CAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACA CCTGAGATCCCAGCCGGGCTC SEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain

TABLE 49 Amino acid sequences of monovalent targeted TnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.14). SEQ ID NO Construct Sequence SEQ ID NO: 136 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWV TnC(18D4) Fc RQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKS hole TSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQG monomeric TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD ligand (71-248) YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV chain TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLV AQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKEL VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPL RSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLS AGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI PAGL SEQ ID NO: 137 Fc knob DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV dimeric ligand TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ (71-248) chain YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK ELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHL SAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTP EIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGM FAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKE DTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLAL HLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF RVTPEIPAGL SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Preparation of Untargeted Split Trimeric 4-1BB Ligand Fc Fusion and Human IgG as Control Molecules

These control molecules were prepared as described above for the TnC targeted construct 6.1 (termed control B), 6.3 (termed control C), 6.4 (termed control D) and 6.5 (termed control E) with the only difference that the anti-TnC binder (VH-VL) was replaced by a germline control, termed DP47, not binding to the antigen).

Table 50 shows, respectively, the cDNA and amino acid sequences of the monovalent DP47-untargeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion containing crossed CH-CL with charged residues, control B.

Table 51 shows, respectively, the cDNA and amino acid sequences of the bivalent DP47-untargeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusion, control C.

Table 52 shows, respectively, the cDNA and amino acid sequences of the monovalent DP47-untargeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross with charged residues, control D.

Table 53 shows, respectively, the cDNA and amino acid sequences of the monovalent DP47-untargeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion without charged residues in the CH-CL cross, control E.

TABLE 50 cDNA and amino acid sequences of monovalent DP47 untargeted split trimeric human 4-1BB ligand (71-254) Fc (kih) fusion with CH-CL cross and with charged residues (control B). SEQ ID NO: Description Sequence SEQ ID NO: 98 nucleotide see above sequence dimeric hu 4- 1BBL (71-254)- CL* Fc knob chain SEQ ID NO: 99 nucleotide see above sequence monomeric hu 4-1BBL (71- 254)-CH1* SEQ ID NO: 138 nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGT sequence DP47 ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG Fc hole chain CCTCCGGATTCACCTTTAGCAGTTATGCCATGAGC TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACA TACTACGCAGACTCCGTGAAGGGCCGGTTCACCAT CTCCAGAGACAATTCCAAGAACACGCTGTATCTGC AGATGAACAGCCTGAGAGCCGAGGACACGGCCGT ATATTACTGTGCGAAAGGCAGCGGATTTGACTACT GGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCT AGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTG CAACGTGAATCACAAGCCCAGCAACACCAAGGTG GACAAGAAAGTTGAGCCCAAATCTTGTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAAGCT GCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA ACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGA AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTC AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA GCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAA AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGC ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA CCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCT ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTG AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA SEQ ID NO: 139 nucleotide GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTC sequence DP47 TTTGTCTCCAGGGGAAAGAGCCACCCTCTCTTGCA light chain GGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCC TGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCT CCTCATCTATGGAGCATCCAGCAGGGCCACTGGCA TCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACA GACTTCACTCTCACCATCAGCAGACTGGAGCCTGA AGATTTTGCAGTGTATTACTGTCAGCAGTATGGTA GCTCACCGCTGACGTTCGGCCAGGGGACCAAAGTG GAAATCAAACGTACGGTGGCTGCACCATCTGTCTT CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCT ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGA TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG TCACAGAGCAGGACAGCAAGGACAGCACCTACAG CCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT ACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGT SEQ ID NO: 102 Dimeric hu 4- see Table 31 1BBL (71-254)- CL* Fc knob chain SEQ ID NO: 103 Monomeric hu see Table 31 4-1BBL (71- 254)-CH1* SEQ ID NO: 140 DP47 Fc hole EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW chain VRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQV SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 141 DP47 light chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC *charges residues

TABLE 51 cDNA and amino acid sequences of bivalent DP47 untargeted split trimeric human 4-1BB ligand (71-254) Fc (kih) fusion (control C). SEQ ID NO: Description Sequence SEQ ID NO: 142 nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGT sequence DP47 ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG Fc hole chain CCTCCGGATTCACCTTTAGCAGTTATGCCATGAGC fused to dimeric TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT hu 4-1BBL (71- GGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACA 254) TACTACGCAGACTCCGTGAAGGGCCGGTTCACCAT CTCCAGAGACAATTCCAAGAACACGCTGTATCTGC AGATGAACAGCCTGAGAGCCGAGGACACGGCCGT ATATTACTGTGCGAAAGGCAGCGGATTTGACTACT GGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCT AGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCC CAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCT CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCC CGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCT CCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGT TCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGT GCCTTCTAGCAGCCTGGGCACCCAGACCTACATCT GCAACGTGAACCACAAGCCCAGCAACACCAAGGT GGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAAGC TGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA AACCCAAGGACACCCTCATGATCTCCCGGACCCCT GAGGTCACATGCGTGGTGGTGGACGTGAGCCACG AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC GGGAGGAGCAGTACAACAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTG CACCCTGCCCCCATCCCGGGATGAGCTGACCAAGA ACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAGCCGGAGAACAACTACAAGACCACGCC TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGT GAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA GGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGG AGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCC GATGATCCTGCTGGACTGCTGGACCTGCGGCAGGG CATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGC TGATCGATGGCCCCCTGTCCTGGTACAGCGATCCT GGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAG CTACAAAGAGGACACCAAAGAACTGGTGGTGGCC AAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGA ACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGC TCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGA AGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGT GGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATA GCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTG TCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACAC AGAGGCCAGGGCTAGACACGCCTGGCAGCTGACA CAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGAC CCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAA GCGAAGGCGGAGGCGGATCTGGCGGCGGAGGATC TAGAGAGGGACCCGAACTGTCCCCTGACGATCCAG CCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCC CAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGG ACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAG GGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAA GATACAAAAGAACTGGTGGTGGCTAAAGCTGGGG TGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGG GTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCT GGCTCTGCATCTGCAGCCACTGCGCTCTGCTGCTG GCGCAGCTGCACTGGCTCTGACTGTGGACCTGCCA CCAGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGG GTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGAC AGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGA GCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAAC TGTGCTGGGACTGTTTCGCGTGACACCTGAGATCC CTGCCGGACTGCCAAGCCCTAGATCAGAA SEQ ID NO: 143 nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGT sequence DP47 ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG Fc knob chain CCTCCGGATTCACCTTTAGCAGTTATGCCATGAGC fused to TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT monomeric hu GGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACA 4-1BBL (71- TACTACGCAGACTCCGTGAAGGGCCGGTTCACCAT 254) CTCCAGAGACAATTCCAAGAACACGCTGTATCTGC AGATGAACAGCCTGAGAGCCGAGGACACGGCCGT ATATTACTGTGCGAAAGGCAGCGGATTTGACTACT GGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCT AGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTG CAACGTGAATCACAAGCCCAGCAACACCAAGGTG GACAAGAAAGTTGAGCCCAAATCTTGTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAAGCT GCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA ACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGA AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTC AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA GCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAA AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAA CCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCT ACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAA CGGCCAGCCTGAGAACAACTACAAGACCACCCCC CCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTA CTCCAAACTGACCGTGGACAAGAGCCGGTGGCAG CAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGA GGCCCTGCACAACCACTACACCCAGAAGTCCCTGA GCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGG AGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCC GATGATCCTGCTGGACTGCTGGACCTGCGGCAGGG CATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGC TGATCGATGGCCCCCTGTCCTGGTACAGCGATCCT GGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAG CTACAAAGAGGACACCAAAGAACTGGTGGTGGCC AAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGA ACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGC TCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGA AGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGT GGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATA GCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTG TCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACAC AGAGGCCAGGGCTAGACACGCCTGGCAGCTGACA CAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGAC CCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAA GCGAA SEQ ID NO: 139 nucleotide See Table 50 sequence DP47 light chain SEQ ID NO: 144 DP47 Fc hole EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW chain fused to VRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR dimeric hu 4- DNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWG 1BBL (71-254) QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQV SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLD LRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEG SGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARN SAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT QGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSRE GPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAAL ALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVH LHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPR SE SEQ ID NO: 145 DP47 Fc knob EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW chain fused to VRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRD monomeric hu NSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYW 4-1BBL (71- GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC 254) LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAG LLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVS LTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVA GEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSE ARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE SEQ ID NO: 141 DP47 light chain See Table 50

TABLE 52 cDNA and amino acid sequences of monovalent DP47 untargeted split trimeric human 4-1BB ligand (71-248) Fc (kih) fusion with CH-CL cross and with charged residues (control D). SEQ ID NO: Description Sequence SEQ ID NO: 114 nucleotide see Table 36 sequence dimeric hu 4- 1BBL (71-248) − CL* Fc knob chain SEQ ID NO: 115 nucleotide see Table 36 sequence monomeric hu 4-1BBL (71- 248) − CH1* SEQ ID NO: 138 nucleotide see Table 50 sequence DP47 Fc hole chain SEQ ID NO: 139 nucleotide see Table 50 sequence DP47 light chain SEQ ID NO: 116 Dimeric hu 4- see Table 37 1BBL (71-248) − CL* Fc knob chain SEQ ID NO: 117 Monomeric hu see Table 37 4-1BBL (71- 248) − CH1* SEQ ID NO: 140 DP47 Fc hole see Table 50 chain SEQ ID NO: 141 DP47 light chain see Table 50 *charged residues

TABLE 53 cDNA and amino acid sequences of monovalent DP47 untargeted split trimeric human 4-1BB ligand (71-248) Fc (kih) fusion with CH-CL cross and without charged residues (control E). SEQ ID NO: Description Sequence SEQ ID NO: 118 nucleotide see Table 38 sequence dimeric hu 4- 1BBL (71-248) − CL Fc knob chain SEQ ID NO: 119 nucleotide see Table 38 sequence monomeric hu 4-1BBL (71- 248) − CH1 SEQ ID NO: 138 nucleotide see Table 50 sequence DP47 Fc hole chain SEQ ID NO: 139 nucleotide see Table 50 sequence DP47 light chain SEQ ID NO: 120 Dimeric hu 4- see Table 39 1BBL (71-248) − CL Fc knob chain SEQ ID NO: 121 Monomeric hu see Table 39 4-1BBL (71- 248) − CH1 SEQ ID NO: 140 DP47 Fc hole see Table 50 chain SEQ ID NO: 141 DP47 light chain see Table 50

Two control human IgG1 molecules containing PGLALA were prepared. Table 54 shows the cDNA and amino acid sequences of germline control DP47 huIgG1 PGLALA (control F). Table 55 shows the cDNA and amino acid sequences of the anti-TnC (18D4) heavy chain (huIgG1 PGLALA, i.e. control L).

TABLE 54 cDNA and amino acid sequences of germline control DP47 huIgG1 PGLALA (control F) SEQ ID NO: Description Sequence SEQ ID NO: 146 nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGT sequence DP47 ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG heavy chain (hu CCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCT IgG1 PGLALA) GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACAT ACTACGCAGACTCCGTGAAGGGCCGGTTCACCATC TCCAGAGACAATTCCAAGAACACGCTGTATCTGCA GATGAACAGCCTGAGAGCCGAGGACACGGCCGTA TATTACTGTGCGAAAGGCAGCGGATTTGACTACTG GGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTA GCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCC TCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCT GGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCA ACGTGAATCACAAGCCCAGCAACACCAAGGTGGA CAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTC ACACATGCCCACCGTGCCCAGCACCTGAAGCTGCA GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC CAAGGACACCCTCATGATCTCCCGGACCCCTGAGG TCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAA AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAG CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA SEQ ID NO: 139 DP47 light chain see Table 50 SEQ ID NO: 147 DP47 heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW chain (hu IgG1 VRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD PGLALA) NSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALG APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQ ID NO: 141 DP47 light chain see Table 50

TABLE 55 cDNA and amino acid sequences of anti-TnC(18D4) heavy chain (huIgG1 PGLALA) (control L) SEQ ID NO: Description Sequence SEQ ID NO: 148 Nucleotide CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAA sequence anti- GAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGG TnC(18D4) CCTCCGGAGGCACATTCAGCAGCTACGCTATAAGC heavy chain TGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTG (huIgG1 GATGGGAGGGATCATCCCTATCTTTGGTACAGCAA PGLALA) ACTACGCACAGAAGTTCCAGGGCAGGGTCACCATT ACTGCAGACAAATCCACGAGCACAGCCTACATGGA GCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGT ATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTG GACTACTGGGGCCAAGGGACCACCGTGACCGTCTC CTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCC TGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCT ACATCTGCAACGTGAATCACAAGCCCAGCAACACC AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGA CAAAACTCACACATGCCCACCGTGCCCAGCACCTG AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGAC CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCATAATGCCAAGACAAAGC CGCGGGAGGAGCAGTACAACAGCACGTACCGTGT GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATCCCGGGATGAGCTGACCAA GAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACGC CTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA SEQ ID NO: 73 Nucleotide see Table 20 sequence 18D4 Light chain SEQ ID NO: 149 anti-TnC(18D4) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW heavy chain VRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITAD (huIgG1 KSTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYW PGLALA) GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 77 18D4 Light see Table 21 chain

Example 9

Production of TnC-Targeted Split Trimeric C-Terminal 4-1BB Ligand Fc Fusion and their Controls

The targeted and untargeted trimeric 4-1BB ligand C-terminal Fc (kih) fusion encoding sequences were cloned into a plasmid vector, which drives expression of the insert from an MPSV promoter and contains a synthetic polyA sequence located at the 3′ end of the CDS. In addition, the vector contains an EBV OriP sequence for episomal maintenance of the plasmid. The split trimeric 4-1BB ligand Fc (kih) fusion molecules were produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using polyethylenimine. The cells were transfected with the corresponding expression vectors. For constructs 6.1, 6.2, 6.4, 6.6 and it's control B, D and E, at a 1:1:1:1 ratio (“vector Fc hole chain”:“vector PD1 light chain”:“vector Fc knob chain”:“vector 4-1BBL light chain”). For constructs 6.3, 6.6, 6.11, 6.12, 6.13, 6.14 and it's control C, at a 1:1:1 ratio (“vector Fc hole chain”:“vector Fc knob chain”:“vector anti-PD1 light chain”). Human IgGs, used as control in the assay, were produced as for the bispecific construct (for transfection only a vector for light and a vector for heavy chain were used at a 1:1 ratio).

For production in 500 mL shake flasks, 300 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 10 minutes at 210×g, and the supernatant was replaced by 20 mL pre-warmed CD CHO medium. Expression vectors (200 μg of total DNA) were mixed in 20 mL CD CHO medium. After addition of 540 μL PEI, the solution was vortexed for 15 seconds and incubated for 10 minutes at room temperature. Afterwards, cells were mixed with the DNA/PEI solution, transferred to a 500 mL shake flask and incubated for 3 hours at 37° C. in an incubator with a 5% CO₂ atmosphere. After the incubation, 160 mL of Excell medium supplemented with 6 mM L-Glutamine, 5 g/L PEPSOY and 1.2 mM valproic acid was added and cells were cultured for 24 hours. One day after transfection 12% Feed were added. After culturing for 7 days, the supernatant was collected by centrifugation for 30-40 minutes at least 400×g. The solution was sterile filtered (0.22 μm filter), supplemented with sodium azide to a final concentration of 0.01% (w/v), and kept at 4° C.

Secreted proteins were purified from cell culture supernatants by affinity chromatography using Protein A, followed by size exclusion chromatography. For affinity chromatography, the supernatant was loaded on a MabSelect Sure column (CV=5-15 mL, resin from GE Healthcare) equilibrated with Sodium Phosphate (20 mM), Sodium Citrate (20 mM) buffer (pH 7.5). Unbound protein was removed by washing with at least 6 column volumes of the same buffer. The bound protein was eluted using either a linear gradient (20 CV) or a step elution (8 CV) with 20 mM sodium citrate, 100 mM Sodium chloride, 100 mM Glycine buffer (pH 2.5). For the linear gradient an additional 4 column volumes step elution was applied.

The pH of collected fractions was adjusted by adding 1/10 (v/v) of 0.5M sodium phosphate, pH8.0. The protein was concentrated prior to loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium chloride, 0.01% (v/v) Tween20 solution of pH 6.0.

The protein concentration was determined by measuring the optical density (OD) at 280 nm, using a molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the targeted trimeric 4-1BB ligand Fc (kih) fusion was analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie SimpleBlue™ SafeStain (Invitrogen USA) or CE-SDS using Caliper LabChip GXII (Perkin Elmer). The aggregate content of samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C.

Table 56 summarizes the yield and final monomer content of the TnC targeted split trimeric 4-1BB ligand Fc (kih) fusion.

TABLE 56 Biochemical analysis of TnC targeted split trimeric 4-1BB ligand Fc (kih) fusion Monomer [%] Yield Construct (SEC) [mg/l] monovalent TnC(18D4) targeted 91.5 3.7 split trimeric 4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL cross without charged residues (construct 6.5) bivalent TnC(18D4) targeted 97 9.9 split trimeric 4-1BB ligand (71-248) Fc (kih) fusion (construct 6.6)

Example 10

Simultaneous Binding of TnC Targeted 4-1BB Split Trimeric Ligand Fc Fusion Antigen Binding Molecule by Surface Plasmon Resonance

The capacity of binding simultaneously human 4-1BB Fc(kih) and human TnC was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

Biotinylated TnC-antigen was directly coupled to a flow cell of a streptavidin (SA) sensor chip. Immobilization levels up to 500 resonance units (RU) were used. The TnC-targeted 4-1BB ligand Fc (kih) fusion molecules were passed at a concentration range of 100 nM with a flow of 30 μL/minute through the flow cells over 90 seconds and dissociation was set to zero sec. Human 4-1BB avi His was injected as second analyte with a flow of 30 μL/minute through the flow cells over 90 seconds at a concentration of 1000 nM (FIGS. 20A and 20B). The dissociation was monitored for 120 sec. Bulk refractive index differences were corrected for by subtracting the response obtained in a reference flow cell, where no protein was immobilized. All bispecific constructs could bind simultaneously human 4-1BB and human TnC (FIGS. 20A and 20B).

Example 11

Affinity Determination of Monovalent TnC Targeted 4-1BB Split Trimeric Ligand Fc Fusion Antigen Binding Molecule by Surface Plasmon Resonance

The binding of TnC-targeted 4-1BB ligand trimer-containing Fc (kih) fusion antigen binding molecules to recombinant TnC was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T100 at 25° C. with HBS-EP as a running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

For affinity measurement, direct coupling of around 500 resonance units (RU) of recombinant human, cynomolgus and murine TnC was performed on a CMS chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). A dilution series (0.05-50 nM) of TnC-targeted 4-1BB ligand trimer-containing Fc (kih) fusion antigen binding molecule was passed on both flow cells at 30 μl/min for 120 sec to record the association phase. The dissociation phase was monitored for 180 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell 1. For the interaction between the TnC-targeted 4-1BB ligand trimer-containing Fc (kih) fusion antigen binding molecule and recombinant TnC, the affinity constants were derived from the rate constants by fitting to a 1:1 Langmuir binding curve using the Biaeval software (GE Healthcare) (Table 57).

TABLE 57 Fittings to 1:1 Langmuir binding and Affinity constants Ligand Analyte ka (1/Ms) kd (1/s) KD (M) monovalent TnC(18D4) targeted Human 2.9E+06 2.0E−03 6.8E−10 split trimeric 4-1BB ligand (71- TnC 248) Fc (kih) fusion containing Cynomolgus 1.5E+06 6.9E−03 4.7E−09 CH-CL cross without charged TnC residues Murine 5.9E+05 2.9E−03 5.0E−09 (construct 6.5) TnC bivalent TnC(18D4) targeted split Human 5.3E+06 1.1E−04 2.1E−11 trimeric 4-1BB ligand (71-248) Fc TnC (kih) fusion Cynomolgus 4.1E+06 1.1E−04 2.7E−11 (construct 6.6) TnC Murine 1.2E+06 3.0E−04 2.5E−10 TnC

Example 12

Functional Characterization of TnC-Targeted 4-1BB Split Trimeric Ligand Fc Fusion Antigen Binding Molecules

A) Binding on Fresh and Activated Human PBMCs of TnC-Targeted Split Trimeric 4-1BB Ligand

Buffy coats were obtained from the Zurich blood donation center. To isolate fresh peripheral blood mononuclear cells (PBMCs) the buffy coat was diluted with the same volume of DPBS (Gibco by Life Technologies, Cat. No. 14190 326). 50 mL polypropylene centrifuge tubes (TPP, Cat.-No. 91050) were supplied with 15 mL Histopaque 1077 (SIGMA Life Science, Cat.-No. 10771, polysucrose and sodium diatrizoate, adjusted to a density of 1.077 g/mL) and the diluted buffy coat contents were layered above the Histopaque 1077. The tubes were centrifuged for 30 min at 450×g. PBMCs were then collected from the interface, washed three times with DPBS and resuspended in T cell medium consisting of RPMI 1640 medium (Gibco by Life Technology, Cat. No. 42401-042) supplied with 10% (v/v) Fetal Bovine Serum (FBS, US-origin, PAN biotech, P30-2009, Lot P150307GI, gamma irradiated mycoplasma free, heat inactivated 35 min 56° C.), 1% (v/v) GlutaMAX I (GIBCO by Life Technologies, Cat. No. 35050 038), 1 mM Sodium-Pyruvat (SIGMA, Cat. No. S8636), 1% (v/v) MEM non-essential amino acids (SIGMA, Cat.-No. M7145) and 50 μM β-Mercaptoethanol (SIGMA, M3148).

PBMCs were used directly after isolation (binding on fresh human PBMCs) or they were stimulated to induce 4-1BB expression on the cell surface of T cells (binding on activated human PBMCs). To induce 4-1BB upregulation on human T cells, PBMCs were cultured for 3 days in RPMI 1640 supplied with 10% FBS, 1 mM Sodium-Pyruvat (SIGMA, Cat.-No. S8636), 1% Gluta-MAX-I, 1% MEM-non essential amino acid Solution (SIGMA, Cat.-No. M7145), 50 μM beta-Mercaptoethanol (Sigma M3148), 200 U/mL Proleukin (Novartis Pharma Schweiz AG) and 2 μg/mL PHA-L (SIGMA, Cat.-No. L2769) to a final concentration of 1×10⁶ cells/mL. After pre-activation cells were harvested and resuspended in RPMI 1640 supplied with 10% PBS, 1 mM Sodium-Pyruvat (SIGMA, Cat.-No. S8636), 1% Gluta-MAX-I, 1% MEM-non essential amino acid solution (SIGMA, Cat.-No. M7145), 50 μM beta-Mercaptoethanol (Sigma M3148) and seeded in 6-well tissue culture plates (TTP, Cat.-No. 92006), which had been coated overnight at 4° C. with 10 μg/mL anti-human CD3 (clone OKT3, Mouse IgG2a, BioLegend, Cat.-No. 317315) and 2 ug/mL anti-human CD28 (clone CD28.2, Mouse IgG1, BioLegend, Cat.-No. 302923). Cells were further incubated for 3 days at 37° C. and 5% CO₂.

For binding assay 0.1×10⁶ fresh or activated PBMCs were added to each well of round-bottom suspension cell 96-well plates (greiner bio-one, cellstar, Cat. No. 650185). Plates were centrifuged 5 minutes with 350×g and at 4° C. and supernatant were discarded. Afterwards cells were stained in 100 μL/well DPBS containing 1:5000 diluted fixable viability dye eF660 (eBioscience, Cat.-No. 65-0864-18) for 30 minutes at 4° C. in the dark. Cells were washed once with 200 μL cold DPBS buffer. Next, 50 μL/well of 4° C. cold FACS buffer (DPBS supplied with 2% FBS, 5 mM EDTA pH8 (Amresco, Cat. No. E177) and 7.5 mM Sodium azide (Sigma-Aldrich, Cat.-No. S2002)) containing titrated concentrations of construct 6.5 and construct 6.6 and as controls control E, control F and control L were added and cells were incubated for 60 minutes at 4° C. and washed three times with 200 μL/well 4° C. FACS buffer. Cells were further resuspended in 50 μL/well of 4° C. cold FACS buffer containing 0.67 μg/mL anti-human CD45-AF488 (clone HI30, mouse igG1k, BioLegend, Cat.-No. 304019), 0.33 μg/mL anti-human CD8a-B V510 (clone SK1, mouse IgG1k, BioLegend, Cat.-No. 344732), 0.23 μg/mL anti-human CD4-BV421 (clone OKT4, mouse IgG2bκ, BioLegend, Cat.-No. 317434), 0.67 μg/mL anti-human CD3-PerCP-Cy5.5 (clone UCHT1, mouse IgG1k, BioLegend, Cat.-No. 300430), 0.67 μg/mL anti-human CD19-PE/Cy7 (clone HIB19, mouse IgG1k, BioLegend, Cat.-No. 302216), 5 μg/mL PE-conjugated AffiniPure anti-human IgG F(ab′)2-fragment-specific goat F(ab′)2 fragment (Jackson ImmunoResearch, Cat. No. 109 116 098) and incubated for 30 min at 4° C. Cells were washed twice with 200 μL/well 4° C. FACS buffer and then fixed with DPBS supplied with 1% Formaldehyde (Sigma, HT501320-9.5L). Cells were resuspended in 100 μL/well FACS-buffer and acquired using the MACS Quant Analyzer 10 (Miltenyi Biotech). Data was analyzed using FlowJo V10 (FlowJo, LLC) and GraphPad Prism 6.04 (GraphPad Software, Inc).

Gates were set on living CD45+ CD3+ CD4+ T cells, CD45+ CD3+ CD8+ T cells, CD45+ CD3+ CD4neg CD8neg γδ T cells and CD45+ CD3neg CD19+ B cells and geo means of fluorescence intensity of PE-conjugated AffiniPure anti-human IgG IgG Fcγ-fragment-specific goat F(ab′)2 fragment were blotted against the titrated concentration of TnC-targeted or DP47-untargeted 4-1BB split trimeric ligand Fc fusion antigen binding molecules and their controls. As shown in FIGS. 21A, 21B, 21C, and 21D, none of the tested TnC-targeted split trimeric 4-1BB ligand fusion molecules or their controls bind to cells of freshly isolated PBMC.

As shown in FIGS. 22A, 22B, 22C, and 22D, after activation living CD45+ CD3+CD4+ T cells, CD45+ CD3+ CD8+ T cells and CD45+ CD3+ CD4neg CD8neg γδ T cells express human 4-1BB. The anti-TnC huIgG1 P329G LALA control L and the DP47 huIgG1 P329G LALA control F did not bind to the activated human PBMCs. All molecules containing a 4-1BB split trimeric ligand as construct 6.5, construct 6.6 and control E bind to activated CD8+ T cells, CD4+ T cells and γδ T cells. In Table 58 the EC50 values are summarized for the binding curves to activated human PBMCs.

TABLE 58 EC50 values of binding curves to activated human PBMCs Control E construct 6.5 construct 6.6 EC50 [nM] on activated CD4 0.23 1.11 8.56 T cells EC50 [nM] on activated CD8 0.26 1.28 1.29 T cells EC50 [nM] on activated γδ 0.23 1.79 0.80 T cells

B) Binding of Human TnC Expressing Tumor Cells

For binding assays on TnC expressing tumor cells the human glioblastoma cell line U87-MG (ATCC HTB-14) was used.

0.2×10⁶ tumor cells resuspended in cold DPBS (Gibco by Life Technologies, Cat. No. 14190 326) were added to each well of a round-bottom suspension cell 96-well plates (greiner bio-one, cellstar, Cat. No. 650185). Cells were washed once with 200 μL DPBS. Cells were resuspended in 100 μL/well of 4° C. cold DPBS buffer containing 1:5000 diluted Fixable Viability Dye eFluor 660 (eBioscience, Cat. No. 65-0864-18) and plates were incubated for 30 minutes at 4° C. Cells were washed once with 200 μL/well 4° C. cold DPBS buffer and resuspended in 50 μL/well of 4° C. cold FACS buffer (DPBS supplied with 2% FBS, 5 mM EDTA pH8 (Amresco, Cat. No. E177) and 7.5 mM Sodium azide (Sigma-Aldrich S2002)) containing TnC-targeted 4-1BB split trimeric ligand Fc fusion antigen binding molecules or their controls at a series of concentrations, followed by incubation for 1 hour at 4° C. After extensive washing, cells were further stained with 50 μL/well of 4° C. cold FACS buffer containing 5 μg/mL PE-conjugated AffiniPure anti-human IgG F(ab′)2-fragment-specific goat F(ab′)2 fragment (Jackson ImmunoResearch, Cat. No. 109 116 098) for 30 minutes at 4° C. Cells were washed twice with 200 μL/well 4° C. FACS buffer and cells were fixed in 50 μL/well DPBS containing 1% Formaldehyde (Sigma, HT501320-9.5L). Cells were resuspended in 100 μL/well FACS-buffer and acquired using the MACS Quant Analyzer 10 (Miltenyi Biotech). Data was analyzed using FlowJo V10 (FlowJo, LLC) and GraphPad Prism 6.04 (GraphPad Software, Inc).

Gates were set on living tumor cells and geo means of fluorescence intensity of PE-conjugated AffiniPure anti-human IgG IgG Fcγ-fragment-specific goat F(ab′)2 fragment were blotted against the titrated concentration of TnC-targeted 4-1BB split trimeric ligand Fc fusion antigen binding molecules or their controls. As shown in FIG. 23 only the TnC-targeted molecules construct 6.5, construct 6.6 and control L bound to TnC-expressing U87-MG whereas the not TnC-targeted controls control E and control F did not bind to the tumor cells. In Table 59 the EC50 values of the curves are listed.

TABLE 59 EC50 values of binding curves to TnC-expressing tumor cells Construct 6.5 Construct 6.6 Control L EC50 [nM] on 73.99 12.75 4.24 U87MG

C) Biological Activity: NF-κB Activation of Human 4-1BB Expressing HeLa Reporter Cell Line

Generation of HeLa Reporter Cells Expressing Human 4-1BB and NF-κB-Luciferase

The human-papilloma-virus-18-induced cervix carcinoma cell line HeLa (ATCC CCL-2) was transduced with a plasmid based on the expression vector pETR10829, which contains the sequence of human 4-1BB (Uniprot accession Q07011) under control of a CMV-promoter and a puromycin resistance gene. Cells were cultured in DMEM-medium (Gibco by Life Technologies Cat. No. 42430-025) supplied with 10% Fetal Bovine Serum (FBS, GIBCO by Life Technologies, Cat. No. 16000-044, Lot. No. 941273, gamma-irradiated mycoplasma free, heat inactivated at 56° C. for 35 minutes), 1% GlutaMAX-I (GIBCO by Life Technologies, Cat.-No. 35050-038) and 3 μg/mL Puromycin (InvivoGen, Cat-No. ant-pr). 4-1BB-transduced HeLa cells were tested for 4-1BB expression by flow cytometry: 0.2×10⁶ living cells were resuspended in 100 μL FACS buffer (DPBS supplied with 2% FBS, 5 mM EDTA pH 8 (Amresco, Cat. No. E177) and 7.5 mM Sodium Azide (Sigma-Aldrich, Cat. No. S2002)) containing 0.1 μg PerCP/Cy5.5 conjugated anti-human 4-1BB mouse IgG1K clone 4B4-1 (BioLegend Cat. No. 309814) or its isotype control (PerCP/Cy5.5 conjugated mouse IgG1K isotype control antibody clone MOPC 21, BioLegend Cat. No. 400150) and incubated for 30 minutes at 4° C. Cells were washed twice with FACS buffer, resuspended in 300 μL FACS buffer containing 0.06 μg DAPI (Santa Cruz Biotec, Cat. No. Sc-3598) and acquired using a 5-laser LSR-Fortessa (BD Bioscience, DIVA software). Limited dilutions were performed to generate single clones as following: Human-4-1BB transduced HeLa cells were resuspended in medium to a concentration of 10, 5 and 2.5 cells/mL and 200 μL, of cell suspensions were transferred to round bottom tissue-culture treated 96-well plates (6 plates/cell concentration, TPP Cat. No. 92697). Single clones were harvested, expanded and tested for 4-1BB expression as described above. The clone with the highest expression of 4-1BB (clone 5) was chosen for subsequent transfection with the NFκB-luciferase expression-vector 5495p Tranlucent HygB. The vector confers transfected cells both with resistance to hygromycin B and capacity to express luciferase under control of NFkB-response element (Panomics, Cat. No. LR0051). Human-4-1BB HeLa clone 5 cells were cultured to 70% confluence. 50 μg (40 μL) linearized (restriction enzymes AseI and SalI) 5495p Tranlucent HygB expression vector were added to a sterile 0.4 cm Gene Pulser/MicroPulser Cuvette (Biorad, Cat.-No, 165-2081). 2.5×10⁶ human-4-1BB HeLa clone 5 cells in 400 μl supplement-free DMEM were added and mixed carefully with the plasmid solution. Transfection of cells was performed using a Gene Pulser Xcell total system (Biorad, Cat No. 165 2660) under the following settings: exponential pulse, capacitance 500 μF, voltage 160 V, resistance ∞. Immediately after the pulse transfected cells were transferred to a tissue culture flask 75 cm² (TPP, Cat. No. 90075) with 15 mL 37° C. warm DMEM-Medium (Gibco by Life Technologies Cat. No. 42430-025) supplied with 10% FBS and 1% GlutaMAX I (GIBCO by Life Technologies, Cat. No. 35050 038). Next day, culture medium containing 3 μg/mL Puromycin (InvivoGen, Cat No. ant pr) and 200 μg/mL Hygromycin B (Roche, Cat. No. 10843555001) was added. Surviving cells were expanded and limited dilution was performed as described above to generate single clones. Clones were tested for 4-1BB expression as described above and for NFκB-Luciferase activity as following: Clones were harvested in selection medium and counted using a Cell Counter Vi-cell xr 2.03 (Beckman Coulter, Cat. No. 731050). Cells were set to a cell density of 0.33×10⁶ cells/mL and 150 μL of this cell suspension were transferred to each well of a sterile white 96-well flat bottom tissue culture plate with lid (greiner bio one, Cat. No. 655083) and as a control to normal 96 well flat bottom tissue culture plate (TPP Cat. No. 92096) to test survival and cell density the next day. Cells were incubated at 37° C. and 5% CO₂ overnight. The next day 50 μL of medium containing different concentrations of recombinant human tumor necrosis factor alpha (rhTNF α, PeproTech, Cat.-No. 300 01A) were added to each well of a 96-well plate resulting in final concentration of rhTNF α of 100, 50, 25, 12.5, 6.25 and 0 ng/well. Cells were incubated for 6 hours at 37° C. and 5% CO₂ and then washed three times with 200 μL/well DPBS. Reporter Lysis Buffer (Promega, Cat-No: E3971) was added to each well (30 μl) and the plates were stored over night at −20° C. The next day frozen cell plates and Detection Buffer (Luciferase 1000 Assay System, Promega, Cat. No. E4550) were thawed to room temperature. 100 uL of detection buffer were added to each well and the plate was measured as fast as possible using a SpectraMax M5/M5e microplate reader and the SoftMax Pro Software (Molecular Devices). Measured URLs above control (no rhTNF-α added) were taken as luciferase activity. The NFκB-luc-4-1BB-HeLa clone 26 was chosen for further use exhibiting the highest luciferase activity and a considerable level of 4-1BB-expression.

NF-κB Activation of HeLa Reporter Cells Expressing Human 4-1BB Co-Cultured with TnC-Expressing U87MG and SkK-Me15 Cells

Adherent human glioblastoma cell line U87MG (ATCC HTB-14) were washed with DPBS (Gibco by Life Technologies, Cat. No. 14190 326) and treated with enzyme-free, PBS-based cell dissociation buffer (Gibco by Life Technologies, Cat.-No. 13151-014) for 10 minutes at 37° C. Afterwards cell were harvested and counted using Cell Counter Vi-cell xr 2.03. Cells were resuspended in DMEM medium (Gibco by Life Technologies Cat. No. 42430 025) supplied with 10% Fetal Calf Serum (FBS, US-origin, PAN biotech, P30-2009, Lot P150307GI, gamma irradiated mycoplasma free, heat inactivated 35 min 56° C.) and 1% GlutaMAX-I (GIBCO by Life Technologies, Cat. No. 35050 038) to 1×10⁶ cells/mL and 100 μL were added/well of sterile white 96-well flat bottom tissue culture plates with lid (greiner bio one, Cat. No. 655083). For negative controls 100 μL medium were added. Plates were incubated overnight at 37° C. and 5% CO₂.

The next day different titrated split trimeric 4-1BB ligand containing fusion molecules and their controls were added. Afterwards NFκB-luc-4-1BB-HeLa clone 26 cells were washed with DPBS and treated with 0.05% Trypsin EDTA (Gibco by Life Technologies Cat. No. 25300 054) for 5 min at 37° C. Cells were harvested and resuspended in DMEM medium supplied with 10% Fetal Calf Serum and 1% GlutaMAX-I. Cells were counted using Cell Counter Vi-cell xr 2.03 (Beckman Coulter, Cat. No. 731050) and set to a cell density of 0.4×10⁶ cells/mL. 50 μL (2×10⁴ cells) of this cell suspension were transferred to each well. Plates were incubated for 6 hours at 37° C. and 5% CO₂. White flat bottom 96-well plates were washed three times with 200 μL/well DPBS. 40 μl fresh prepared Reporter Lysis Buffer (Promega, Cat-No: E3971) were added to each well and the plate were stored over night at −20° C. The next day frozen plates and detection buffer (Luciferase 1000 Assay System, Promega, Cat. No. E4550) were thawed to room temperature. 100 uL of detection buffer were added to each well and plates were measured as fast as possible using the SpectraMax M5/M5e microplate reader and SoftMax Pro Software (Molecular Devices) with following settings: for luciferase (RLUs), 500 ms integration time, no filter, collecting all wave length and top reading.

TABLE 60 EC50 values of NFκB-activation- induced Luciferase activity-curves construct 6.5 construct 6.6 U87MG EC50 [nM] 0.33 0.70

D) Activation Assay of Human PBMCs in the Presence of TnC-Expressing Tumor Cells

For TnC-binding-mediated crosslinking the TnC-expressing adherent human glioblastoma cell line U87MG (ATCC HTB-14) was used. U87MG cells were washed with DPBS (Gibco by Life Technologies, Cat. No. 14190 326) and treated with enzyme-free, PBS-based Cell Dissociation Buffer (Gibco by Life Technologies, Cat.-No. 13151-014) for 10 minutes at 37° C. Cells were harvested and resuspended in T cell medium consisting of RPMI 1640 (GIBCO by Life Technologies, Cat.-No. 42401-042) supplied with 10% fetal bovine serum (FBS, US-origin, PAN biotech, P30-2009, Lot P150307GI, gamma irradiated mycoplasma free, heat inactivated 35 min 56° C.), 1% L-GlutaMAX-I (GIBCO by Life Technologies, Cat-No. 35050-038), 1 mM Sodium-Pyruvat (SIGMA-Aldrich, Cat.-No. S8636), 1% MEM-Non essential Aminoacid Solution (SIGMA-Aldrich, Cat.-No. M7145), 50 μM β-Mercaptoethanol (Sigma-Aldrich, Cyt.-No. M3148) and irradiated with 50 Gy (X-Ray Irradiator RS 2000, Rad source). 2×10⁴ U87MG cells in 50 μL T cell medium were seeded to each well of a round bottom tissue culture 96-well plate (TTP, Cat.-No. 92697). 50 μL, of T cell medium containing different titrated concentrations of construct 6.5 and 6.6 or the fitting controls (control F, control L and control E) were added. Human PBMCs were labeled in 37° C. warm DPBS containing 40 nM CFDA-SE (SIGMA-Aldrich, Cat.-No. 21888-25MG-F) for 15 min at 37° C. CFSE-labeling was stopped by adding FBS, PBMCs were washed twice and resuspended in T cell medium to a final concentration of 1.5×10⁶ cells/mL. 50 μL of this PBMC cell solution were seeded to each well e.g. 7.5×10⁴ CFSE-labeled PBMCs were added to each well. Finally a stock solution of T cell medium containing 8 nM agonistic anti-human CD3 human IgG1 clone V9 was prepared and 50 μL/well were added to each well giving a final concentration of 2 nM anti-human CD3 human IgG1 clone V9.

Plates were incubated for 4 days at 37° C. Cells were washed with DPBS and stained with 100 μL/well DPBS containing 1:1000 diluted LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Molecular Probes by Life Technology, Cat.-No. L34957) for 30 min at 4° C. Cells were washed once with 200 μL/well DPBS and stained with 50 μL FACS buffer (DPBS supplied with 2% FBS, 5 mM EDTA pH8 (Amresco, Cat. No. E177) and 7.5 mM Sodium azide (Sigma-Aldrich S2002)) containing 0.1 μg/mL PerCP-Cy5.5-conjugated anti-human CD137 mouse IgG1 κ (clone 4B4-1, BioLegend, Cat.-No. 309814), 0.1 μg/mL PE/Cy7-conjugated anti-human PD-1 mouse IgG1 κ (clone EH12.2H7, BioLegend, Cat.-No. 329918), 0.03 μg/mL APC-conjugated anti-human CD25 mouse IgG1 κ (clone BC96, BioLegend, Cat.-No. 302610), 0.06 μg/mL APC/Cy7-conjugated anti-human CD8 Mouse IgG1 κ (clone RPA-T8, BioLegend, Cat.-No. 3301016), BV421-conjugated anti-human CD4 Mouse IgG1 κ (clone RPA-T4, BioLegend, Cat.-No. 300532) for 30 min at 4° C. Cells were washed twice with 200 μL/well DPBS and incubated for 30 min at 4° C. with 50 μL/well freshly prepared FoxP3 Perm/Fix buffer (eBioscience Cat.-No. 00-5123). Cells were washed twice with 200 μL/well DPBS, resuspended in 50 μL/well freshly prepared Perm-buffer (eBioscience Cat.-No 00-8333) supplied with PE-conjugated 1:250 diluted anti-human Granzyme B mouse IgG1 κ (clone GB11, Lot 4269803, BD Pharmingen, Cat.-No. 561142) and incubated for 1 h at 4° C. Plates were washed twice with 200 μL/well DPBS and cells were fixed for 15 min with DPBS containing 1% Formaldehyde (Sigma, HT501320-9.5L). Cells were resuspended in 100 μL/well FACS-buffer and acquired using the MACS Quant Analyzer 10 (Miltenyi Biotech). Data was analyzed using FlowJo V10 (FlowJo, LLC) and GraphPad Prism 6.04 (GraphPad Software, Inc).

As shown in FIGS. 27A and 27B only the TnC-targeted split trimeric 4-1BBL fusion molecules construct 6.6 and to a less extent construct 6.5 induced a concentration-dependent increased surface expressed of CD25 and 4-1BB on CD8+ T cells. The negative control molecules (control F, control L and control E) did not induce this upregulation of activation markers. The correlating EC50 values are listed in Table 61.

TABLE 61 EC50 values of upregulation of CD25 and 4-1BB (CD137) on activated CD8+ T cells Construct 6.5 Construct 6.6 % CD25+ CD8 EC50 [nM] 0.13 0.29 % CD137 (4-1BB)+ CD8 EC50 [nM] 0.36 0.13

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The invention claimed is:
 1. A TNF (tumor necrosis factor) family ligand trimer-containing antigen binding molecule comprising (a) at least one antigen binding moiety capable of specific binding to Tenascin-C(TnC) and (b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or a fragment thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof.
 2. The TNF family ligand trimer-containing antigen binding molecule of claim 1, further comprising (c) an Fc domain composed of a first and a second subunit capable of stable association.
 3. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the antigen binding molecule is further characterized in that (i) the first polypeptide contains a CH1 or CL domain and the second polypeptide contains a CL or CH1 domain, respectively, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domain, or (ii) the first polypeptide contains a CH3 domain and the second polypeptide contains a CH3 domain, respectively, or (iii) the first polypeptide contains a VH-CL or a VL-CH1 domain and the second polypeptide contains a VL-CH1 domain or a VH-CL domain, respectively, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domain.
 4. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the TNF ligand family member costimulates human T-cell activation.
 5. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the TNF ligand family member is selected from 4-1BBL and OX40L.
 6. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the TNF ligand family member is 4-1BBL.
 7. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the moiety capable of specific binding to TnC is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, an aVH and a scaffold antigen binding protein.
 8. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the molecule comprises one or two moieties capable of specific binding to TnC.
 9. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the antigen binding moiety capable of specific binding to TnC is a Fab molecule capable of specific binding to TnC.
 10. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the antigen binding moiety capable of specific binding to TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:
 57. 11. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the antigen binding moiety capable of specific binding to TnC comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 71, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 59, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:
 60. 12. The TNF family ligand trimer-containing antigen binding molecule of claim 2, wherein the Fc domain is an IgG1 Fc domain comprising the amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering).
 13. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the antigen binding molecule comprises a first heavy chain and a first light chain, both comprising a Fab molecule capable of specific binding to TnC, a first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker fused at its C-terminus by a second peptide linker to a second heavy or light chain, and a second peptide comprising one ectodomain of said TNF ligand family member fused at its C-terminus by a third peptide linker to a second light or heavy chain, respectively.
 14. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a CH1 domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a CL domain that is part of a light chain.
 15. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a CL domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a CH1 domain that is part of a light chain.
 16. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the first peptide comprising two ectodomains of a TNF ligand family member or fragments thereof connected to each other by a first peptide linker is fused at its C-terminus by a second peptide linker to a VH domain that is part of a heavy chain, and the second peptide comprising one ectodomain of said TNF ligand family member or a fragment thereof is fused at its C-terminus by a third peptide linker to a VL domain that is part of a light chain.
 17. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the antigen binding moiety binds to human TnC, and cross-reacts to mouse and cynomolgus TnC.
 18. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the antigen binding moiety binds to at least one of human, mouse and cynomolgus TnC with a K_(D) range of about 1 μM to about 0.001 nM.
 19. The TNF family ligand trimer-containing antigen binding molecule of claim 17, wherein the antigen binding moiety binds to human TnC with a K_(D) range of about 1 μM to about 0.001 nM, and cross-reacts to mouse and cynomolgus TnC with a binding affinity within the K_(D) range of a factor of 20 relative to the K_(D) for binding human TnC.
 20. The TNF family ligand trimer-containing antigen binding molecule of claim 1, for use as a medicament.
 21. The TNF family ligand trimer-containing antigen binding molecule of claim 1, for use in the treatment of cancer.
 22. The TNF family ligand trimer-containing antigen binding molecule of claim 1, wherein the antigen binding moiety binds to human TnC with a K_(D) range of about 1 μM to about 0.001 nM, and cross-reacts to mouse and cynomolgus TnC with a binding affinity within the K_(D) range of a factor of 20 relative to the K_(D) for binding human TnC.
 23. A pharmaceutical composition comprising the TNF family ligand trimer-containing antigen binding molecule of and at least one pharmaceutically acceptable excipient wherein the TNF family ligand trimer-containing antigen binding molecule comprises: (a) at least one antigen binding moiety capable of specific binding to Tenascin-C(TnC) and (b) a first and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or a fragment thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof.
 24. The TNF family ligand trimer-containing antigen binding molecule of the pharmaceutical composition of claim 23, for use as a medicament.
 25. The pharmaceutical composition of claim 23 for use in the treatment of cancer. 